Fruit-specific promoters

ABSTRACT

The present disclosure provides genetic constructs containing a promoter that is useful in driving fruit-specific expression in plants. Further provided are expression vectors, transgenic plants and plant parts containing such genetic constructs, as well as uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/675,637, filed May 23, 2018, which is hereby incorporated byreference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 757682000100SEQLIST.TXT,date recorded: May 23, 2019, size: 19 KB).

FIELD OF THE DISCLOSURE

The present disclosure relates generally to the field of plant breedingand biotechnology. More specifically, it relates to fruit-specificpromoters and uses thereof.

BACKGROUND

Fruits are an important source of nutrients, minerals, vitamins, anddietary fiber, and as such, significant efforts have been made to breedfor fruits with higher yield and better quality. Traditional methods offruit breeding have been hampered by a number of challenges, includinglarge size of the plant, long juvenile phase, and limited genetic gains.Recently, advances in genomics and genetic engineering have been used togenetically improve fruit crops by affording new sources ofcharacteristics and shortened breeding cycles. Genetic engineering offruit crops involves directly manipulating the genome of a fruitspecies, typically by introducing into the genome a genetic constructcarrying exogenous genetic information, e.g., a foreign gene. Oncesuccessfully integrated into the host genome, the exogenous geneticinformation may express and result in a favorable change in thephysiological and morphological properties of the organism, leading toan improvement of the desired trait.

To express exogenous genetic information in the host organism, thegenetic construct needs to contain certain regulatory sequences, such asa promoter. During the various stages of the growth and development ofthe plant, and as to the various parts and organs of the plant, it isoften desirable to direct the effect of the genetic construct to aparticular plant part and/or to a particular growth stage, such that theexogenous genetic information is expressed with minimum adverse sideeffects on normal plant growth and development. Accordingly, it would beadvantageous for the promoter of a genetic construct to be able todirect transgene expression in the appropriate cell types(tissue-specific expression) and/or at the appropriate time indevelopment (development-specific expression). Therefore, promoters thatare capable of driving fruit-specific expression would be valuablegenetic tools for engineering fruit traits of agronomic importance, suchas growth, ripening, nutritional quality, and post-harvest shelf life.

To date, a number of fruit-specific promoters have been isolated andcharacterized from various plant species, mostly from tomato. Forinstance, the tomato E4 and E8 promoters have been found to befruit-specific that are coordinately regulated by ethylene during fruitripening (Deikman, et al. (1992) Plant Physiol. 100:2013-2017; Xu et al.(1996) Plant Mol. Biol. 31: 1117-1127). Additional tomato fruit-specificpromoters include the promoter of the polygalacturonase gene (PG), whichplays a role in cell wall degradation during fruit ripening (Bird et al.(1988) Plant Mol. Biol. 11:651-662; Lau et al (2009) Plant Mol. Biol.Rep. 27:250-256); the promoter of the T-proline-rich protein F1(TPRP-F1) gene, which is specifically expressed in the ovary and youngfruit (Salts et al. (1992) Plant Mol. Biol. 18:407-409); and thepromoter of ACC synthase (Lin et al. (2007) Chin. Sci. Bull.52:1217-1222). A few fruit-specific promoters have also been isolatedfrom non-tomato plant species, such as the promoter of theripening-upregulated gene ACC-oxidase in apple and peach, the promoterof PG from apple, and the promoter of expansin from sour cherry(Atkinson et al. (1998) Plant Mol. Biol. 38:449-460; Rasori et al.(2003) Plant Sci. 165:523-530). However, in many fruit species theavailability of fruit-specific promoters suitable for use in geneticengineering is still limited.

Global demand for fruit products is continuously on the rise. Thus, aneed exists for the identification of novel promoters providing robustand reliable fruit-specific expression and for their use in theengineering of fruit crops towards improved agronomic traits andnutritional quality.

SUMMARY

In order to meet the above and other needs, the present disclosureprovides genetic constructs containing a promoter driving fruit-specificexpression in plants. The present disclosure also provides expressionvectors, transgenic plants and plant parts containing the geneticconstructs described herein. Further provided are methods of using thedisclosed genetic constructs in fruit breeding.

Accordingly, one aspect of the present disclosure relates to a geneticconstruct containing a promoter operably linked to a heterologousnucleotide sequence encoding a product of interest, where the promotercomprises a sequence selected from SEQ ID NOs: 1-5, or a sequence havingat least 90% identity thereto. In some embodiments, the product ofinterest is an RNA molecule. In some embodiments, the product ofinterest is a polypeptide. In some embodiments, the product of interestis in an anthocyanin metabolic pathway, in a tocopherol metabolicpathway, in a fatty acid metabolic pathway, in a carotenoid metabolicpathway, in a lycopene metabolic pathway, in a betalain metabolicpathway, and/or in a flavonoid metabolic pathway. In some embodiments,the product of interest is a MYB transcription factor, a phytoenesynthase (PSY), a lycopene cyclase (LCY), or a DXP synthase (DXS).

Another aspect of the present disclosure relates to expression vectors,transgenic plants and plant parts containing a genetic construct of anyof the preceding embodiments. In some embodiments, the presentdisclosure relates to an expression vector having a genetic construct ofany of the preceding embodiments. In some embodiments, the presentdisclosure relates to a transgenic plant having a genetic construct ofany of the preceding embodiments. In some embodiments, the presentdisclosure relates to a plant part of the transgenic plant, where theplant part contains a genetic construct of any of the precedingembodiments. In some embodiments, the plant part is a stem, a branch, aroot, a leaf, a flower, a fruit, a seed, a cutting, a bud, a cell, or aportion thereof.

Yet another aspect of the present disclosure includes a method ofmodifying a fruit phenotype in a plant, the method having the steps of:i) transforming a plant cell with a genetic construct of any of thepreceding embodiments, where expression of the product of interest isassociated with modification of the fruit phenotype; ii) regenerating aplant from the transformed plant cell; and iii) growing the regeneratedplant to produce fruit of the modified phenotype. In some embodiments,the fruit phenotype is selected from size, weight, color, shape,firmness, glossiness, flavor, aroma, secondary metabolite content, peelthickness, seed number, juice quality, juice sugar content, juice acidcontent, juice taste, juice color, and juice yield. In some embodiments,the fruit phenotype is selected from anthocyanin content, tocopherolcontent, fatty acid content, carotenoid content, lycopene content,betalain content, and flavonoid content.

In some embodiments that may be combined with any of the precedingembodiments, the plant is selected from orange (Citrus sinensis),mandarin (Citrus reticulata), lime (Citrus aurantifolia), grapefruit(Citrus paradisi), lemon (Citrus limon), pomelo (Citrus maxima), citron(Citrus medico), papeda (Citrus micrantha), and Prunus sp.

The present disclosure is based, at least in part, on the unexpecteddiscovery that tomato plants expressing a genetic construct describedherein, where the genetic construct comprises a promoter sequence of SEQID NO: 4 or a sequence having at least 90% identity thereto, produceseedless fruit. Accordingly, another aspect of the present disclosureprovides a method of creating a tomato plant with seedless fruit, themethod having the steps of 1) transforming a tomato plant cell with agenetic construct described herein, where the promoter comprises thesequence of SEQ ID NO: 4, or a sequence having at least 90% identitythereto; ii) regenerating a tomato plant from the transformed tomatoplant cell; and iii) growing the regenerated tomato plant to produceseedless fruit.

It is to be understood that one, some, or all of the properties of thevarious embodiments described above and herein may be combined to formother embodiments of the present disclosure. These and other aspects ofthe present disclosure will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows the expression patterns of tissue specificity for thecandidate fruit-specific citrus genes #1-#4.

FIG. 2 shows the genetic constructs containing selected candidatefruit-specific promoters from citrus (CitSEPp, CitWAXp, CitUNKp,CitJuSacp) and plum (PfeMybAp), as well as fruit-specific promoters fromtomato (E8p and PGp) and constitutive promoter 35Sp as controls.

FIGS. 3A-3E show the various sequence elements identified in thecandidate fruit-specific promoters. FIG. 3A shows the various sequenceelements identified in the candidate fruit-specific promoter CitSEPp.FIG. 3B shows the various sequence elements identified in the candidatefruit-specific promoter CitWAXp. FIG. 3C shows the various sequenceelements identified in the candidate fruit-specific promoter CitUNKp.FIG. 3D shows the various sequence elements identified in the candidatefruit-specific promoter CitJuSacp. FIG. 3E shows the various sequenceelements identified in the candidate fruit-specific promoter PfeMybAp.

FIG. 4 shows the results of the Agrobacterium-mediated transientexpression assay for testing the functionality of the candidatefruit-specific Promoter::GUS constructs in unripe (left) and ripe(right) tomato fruits as compared to control (wide type tomatoAgroinjected with empty vector).

FIG. 5 shows the phenotypes of the transgenic tomato plants stablytransformed with various Promoter::GUS constructs.

FIG. 6 shows the seedless fruit phenotype in the transgenic CitJuSacptomato plants as compared to wild type.

FIG. 7 shows the comparison of fruit phenotype (upper panel) andcomparison of genome DNA content (lower panel) between the transgenicCitJuSacp tomato lines and wild type.

FIGS. 8A-8B show GUS staining on whole seedling (column 1), mature leaf(column 2), flower (column 3), unripe fruit cross section (column 4),unripe fruit exterior (column 5), ripe fruit cross section (column 6),and ripe fruit exterior (column 7) in wild type tomato versus intransgenic tomato lines transformed with the Promoter::GUS constructs(35Sp::GUS, E8p::GUS, PGp::GUS, CitSEPp::GUS and CitWAXp::GUS constructsin FIG. 8A; CitUNKp::GUS, CitJuSacp::GUS and PfeMybAp::GUS constructs inFIG. 8B). Representative plant tissues are from wild type tomato(cultivar Micro-Tom) or transgenic plants after one month of cultivationin the greenhouse.

FIGS. 9A-9D show the quantitative GUS analysis on leaf (L), unripe fruit(UR) and ripe fruit (R) in representative transgenic tomato linestransformed with the Promoter::GUS constructs as compared to wild type.FIG. 9A shows the quantitative GUS analysis on leaf (L) inrepresentative transgenic tomato lines transformed with thePromoter::GUS constructs as compared to wild type. FIG. 9B shows thequantitative GUS analysis on unripe fruit (UR) in representativetransgenic tomato lines transformed with the Promoter::GUS constructs ascompared to wild type. FIG. 9C shows the quantitative GUS analysis onripe fruit (R) in representative transgenic tomato lines transformedwith the Promoter::GUS constructs as compared to wild type. FIG. 9Dshows the quantitative GUS analysis on leaf (L), unripe fruit (UR) andripe fruit (R) in representative transgenic tomato lines transformedwith the CitSEPp::GUS construct as compared to wild type.

FIG. 10 shows the GUS staining results in transgenic Arabidopsis,tobacco and tomato plants transformed with the CitSEPp::GUS construct.

FIG. 11 shows the molecular constructs used for the anthocyaninaccumulation study. For generating transgenic Arabidopsis and tobaccoplants, the Promoter::MoroMybA constructs were inserted into thepCTAG6-GUSPlus vector backbone for transformation (upper panel). Forgenerating transgenic citrus plants, the Promoter::MoroMybA constructsas well as the Nosp::AtFT::NosT construct were inserted into thepCTAG6-GUSPlus vector backbone for transformation (lower panel). Nosp,nopaline synthase promoter; AtFT, Arabidopsis FLOWERING LOCUS T gene;NosT, nopaline synthase terminator.

FIG. 12 shows the phenotypes of transgenic tobacco plants transformedwith the 35Sp::MoroMybA construct as compared to wild type.

FIG. 13 shows the phenotypes of transgenic Arabidopsis plantstransformed with the Promoter::MoroMybA constructs as compared to wildtype.

FIG. 14 shows the phenotypes of transgenic tobacco plants transformedwith the Promoter::MoroMybA constructs.

FIGS. 15A-15E show the results of semi-quantitative reversetranscription PCR (RT-PCR) for transgenic citrus plants transformed withthe Promoter::MoroMybA constructs as compared to wild type. The upperpanel in each figure shows in histograms the AtFT expression relative tothe internal control CitEF1a (Y-axis) on transgenic citrus lines withdifferent copy numbers (CN, X-axis) and wild type for each constructtested. The bars highlighted in pink are the early-flowering lines. Thelower panel in each figure shows the corresponding agarose gelelectrophoresis results. FIG. 15A shows the results of RT-PCR fortransgenic citrus plants transformed with the CitWAXp::MoroMybAconstruct as compared to wild type. FIG. 15B shows the results of RT-PCRfor transgenic citrus plants transformed with the CitUNKp::MoroMybAconstruct as compared to wild type. FIG. 15C shows the results of RT-PCRfor transgenic citrus plants transformed with the CitJuSacp::MoroMybAconstruct as compared to wild type. FIG. 15D shows the results of RT-PCRfor transgenic citrus plants transformed with the PfeMybAp::MoroMybAconstruct as compared to wild type. FIG. 15E shows the results of RT-PCRfor transgenic citrus plants transformed with the E8p::MoroMybAconstruct as compared to wild type.

FIG. 16 shows the phenotypes of unripe fruit and flower of thetransgenic citrus plants transformed with the Promoter::MoroMybAconstructs as compared to wild type.

FIG. 17 shows the phenotypes of the transgenic citrus plant line #9-3transformed with the CitWAXp::MoroMybA construct.

FIG. 18 shows the phenotypes of the transgenic citrus plant line #4-3transformed with the CitUNKp::MoroMybA construct.

FIG. 19 shows the phenotypes of the transgenic citrus plant line #7-19transformed with the CitJuSacp::MoroMybA construct. Purple colorationindicating signs of anthocyanin accumulation in young plants ishighlighted in black circle.

FIG. 20 shows the phenotypes of the transgenic citrus plant line #5-1transformed with the PfeMybAp::MoroMybA construct.

FIG. 21 shows the phenotypes of the transgenic citrus plant line #10-11transformed with the E8p::MoroMybA construct.

FIG. 22 shows that the transgenic Arabidopsis lines trasnformed with the35S::PSY construct accumulated lycopene only in the siliques, giving anorange coloration (marked in black circles).

FIG. 23 shows the phenotypes of the initial transgenic Carrizo testlines transformed with the 35S::β/ε cyclase RNAi::PSY construct.

FIG. 24 shows the CitWAXp::RNAi ε-&β-LCY-CitJuSacp::PSY-CitUNKp::DXSconstruct and the FT (flowering gene) construct used in the transgeniccitrus lycopene accumulation study.

FIG. 25 shows the phenotype of lycopene accumulation in the transgenicMexican lime callus transformed with the CitWAXp::RNAiε-&β-LCY-CitJuSacp::PSY-CitUNKp::DXS construct and the FT (floweringgene) construct, with the signs of lycopene accumulation as light orangeblush marked in black circle.

FIG. 26 shows the transgenic Mexican lime (upper panel) and Carrizo(lower panel) transformed with the CitWAXp::RNAiε-&β-LCY-CitJuSacp::PSY-CitUNKp::DXS construct and the FT (floweringgene) construct, with no visible lycopene accumulation in the vegetativegrowth of the plants.

DETAILED DESCRIPTION

The following description is presented to enable a person of ordinaryskill in the art to make and use the various embodiments. Descriptionsof specific materials, techniques, and applications are provided only asexamples. Various modifications to the examples described herein will bereadily apparent to those of ordinary skill in the art, and the generalprinciples defined herein may be applied to other examples andapplications without departing from the spirit and scope of the variousembodiments. Thus, the various embodiments are not intended to belimited to the examples described herein and shown, but are to beaccorded the broadest scope consistent with the claims.

The present disclosure relates generally to genetic constructscontaining a promoter allowing for tissue-specific expression in plants.Further embodiments relate generally to expression vectors, transgenicplants and transgenic plant parts containing a genetic constructdisclosed herein, as well as methods of use thereof.

Genetic Constructs of the Disclosure

In one aspect, the present disclosure provides a genetic constructcontaining a promoter operably linked to a heterologous nucleotidesequence encoding a product of interest, where the promoter comprises asequence selected from SEQ ID NOs: 1-5, or a sequence having at least90% identity thereto.

As used herein, a “genetic construct” or “construct” refers to anartificially assembled nucleic acid molecule containing one or moregenetic elements in a deliberately arranged order. A genetic constructmay be generated from any type of nucleic acid, e.g., DNA, RNA orvariants thereof. For instance, a genetic construct that is artificiallyassembled from DNA is referred to as a “DNA construct”. A geneticconstruct may also contain any type of genetic element, including butnot limited to, an enhancer, a silencer, a promoter, a 5′ untranslatedregion (5′ UTR), an open reading frame (ORF), an exon, an intron, aprotein-coding region, a functional RNA-coding region, a 3′ untranslatedregion (3′ UTR), a terminator, and fragments thereof.

In some embodiments, the genetic construct of the present disclosure isan expression construct. As used herein, an “expression construct” is agenetic construct that contains necessary genetic elements that arepositioned in a way capable of conferring transcription and/ortranslation in a host cell. As used herein, the term “transcription”refers to the synthesis of RNA molecules from DNA templates, and theterm “translation” refers to the synthesis of polypeptide molecules fromRNA templates. As used herein, the term “expression” or “geneexpression” refers to transcription of a DNA template (e.g., gene,genetic construct) into RNA (e.g., mRNA, tRNA, rRNA, non-coding RNA),with or without subsequent translation of the RNA into a polypeptide.Expression may include both transcription and translation, as well asany modification and processing of the products therein.

As used herein, “expression pattern” of a gene or genetic constructrelates to its being transcribed at a certain time during plant growthand development (temporal expression pattern), and/or in a certainlocation in the plant (spatial expression pattern). It may be desirablefor a gene or genetic construct to have a particular expression patternto achieve optimal effect. For example, constitutive expression of agene product may be beneficial in one location of the plant but lessbeneficial in another part of the plant; in other cases, it may bebeneficial to have a gene product produced at a certain developmentalstage of the plant or in response to certain environmental or chemicalstimuli. As used herein, the terms “tissue-specific expression”,“tissue-preferred expression” and “tissue-preferential expression” areused interchangeably to refer to a pattern of expression that issubstantially limited to certain tissue types. Tissue-specificexpression is not necessarily limited to expression in a single tissuebut may include expression limited to one or more specific tissues, suchas the specific tissues within one organ. For example, an expressionpattern that is substantially limited to the specific tissues within afruit is referred to as tissue-specific expression in fruit. As usedherein, the terms “tissue-specific expression in fruit” and“fruit-specific expression” are used interchangeably and refer toexpression that is substantially limited to fruit of a plant.

Promoters

As used herein, the term “promoter” refers to a nucleotide sequencecapable of controlling the expression of a particular nucleotidesequence of interest. A promoter may include one or more promoterelements. As used herein, a “promoter element” or “cis-element” means anelement that influences the characteristics and/or activities of thepromoter, such as temporal and spatial expression patterns. Examples ofa promoter element include, without limitation, TATA box, CAAT box, GCbox, and CAP site. Methods of identifying promoter elements are wellknown in the art. For example, a promoter sequence may be analyzed forknown promoter elements using tools such as Plant Promoter AnalysisNavigator (PlantPAN, see Chang et al. (2008) BMC Genomics 9(1):561-561),PLAnt Cis-acting regulatory DNA Elements (PLACE, see Higo et al. (1999)Nucleic Acids Research 27(1):297-300), and Plant Cis-acting RegulatoryElements (PlantCARE, see Lescot et al. (2002) Nucleic Acids Research30(1):325-327).

In one aspect, the present disclosure provides promoters that arecapable of directing tissue-specific expression in fruit. In someembodiments, the promoter comprises a nucleotide sequence of SEQ IDNO: 1. In some embodiments, the promoter comprises a nucleotide sequenceof SEQ ID NO: 2. In some embodiments, the promoter comprises anucleotide sequence of SEQ ID NO: 3. In some embodiments, the promotercomprises a nucleotide sequence of SEQ ID NO: 4. In some embodiments,the promoter comprises a nucleotide sequence of SEQ ID NO: 5. As usedherein, the terms “fruit-specific”, “fruit-preferred”, and“fruit-preferential” are used interchangeably to refer to a pattern ofexpression that is predominantly in fruit of a plant.

Homologs, orthologs, and paralogs of the promoter of the presentdisclosure may be identified by sequence identify and isolated usingmethods known in the art. As used herein, the term “sequence identity”refers to the state of having identical residues in the same locationswhen two or more nucleic acid or amino acid sequences are aligned. Insome embodiments, the promoter of the present disclosure comprises anucleotide sequence having a certain degree of sequence identify to anyone of the SEQ ID NOs: 1-5. The term “% identical” or “% identity” asused herein, refers to the percentage or level of nucleotide or aminoacid sequence identity between two or more aligned sequences. Thedetermination of percent sequence identity and/or similarity between anytwo sequences may be accomplished using a mathematical algorithm.Examples of such mathematical algorithms are the algorithm of Myers andMiller, CABIOS 4:11-17 (1988); the local homology algorithm of Smith etal., Adv. Appl. Math. 2:482 (1981); the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); thesearch-for-similarity-method of Pearson and Lipman, Proc. Natl. Acad.Sci. 85:2444-2448 (1988); the algorithm of Karlin and Altschul, Proc.Natl. Acad. Sci. USA 87:2264-2268 (1990), modified as in Karlin andAltschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993). Computerimplementations of these mathematical algorithms can be utilized forcomparison of sequences to determine sequence identity and/orsimilarity. Such implementations include, for example: CLUSTAL in thePC/Gene program (Intelligenetics, Mountain View, Calif.); the AlignXprogram, version10.3.0 (Invitrogen, Carlsbad, Calif.) and GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Version 8 (Genetics Computer Group, Madison, Wis.). Alignments usingthese programs can be performed using the default parameters. TheCLUSTAL program is well described by Higgins et al. Gene 73:237-244(1988); Higgins et al. CABIOS 5:151-153 (1989); Corpet et al., NucleicAcids Res. 16:10881-90 (1988); Huang et al. CABIOS 8:155-65 (1992); andPearson et al., Meth. Mol. Biol. 24:307-331 (1994). The BLAST programsof Altschul et al. J. Mol. Biol. 215:403-410 (1990) are based on thealgorithm of Karlin and Altschul (1990) supra.

Thus, accordingly, in some embodiments, the promoter of the presentdisclosure comprises a nucleotide sequence that is at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%identical to SEQ ID NO: 1. In some embodiments, the promoter of thepresent disclosure has a nucleotide sequence that is at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%identical to SEQ ID NO: 2. In some embodiments, the promoter of thepresent disclosure has a nucleotide sequence that is at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%identical to SEQ ID NO: 3. In some embodiments, the promoter of thepresent disclosure has a nucleotide sequence that is at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%identical to SEQ ID NO: 4. In some embodiments, the promoter of thepresent disclosure has a nucleotide sequence that is at least about 50%,at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, at least about 99%, or 100%identical to SEQ ID NO: 5.

A plant promoter typically comprises a core promoter, and often,additional regulatory elements. The core promoter is the minimalsequence that is required for directing basal level of expression,comprising a TATA box region where RNA polymerase, TATA-binding protein(TBP), and TBP-associated factors may bind to initiate transcription. Inaddition to the core promoter, further sequence elements are oftennecessary for initiating transcription that has a tissue- ordevelopmental stage-specific expression pattern. For instance, theTGTCACA motif has been found to be a cis-regulatory enhancer elementnecessary for fruit-specific expression in melon species (Yamagata etal. (2002) J. Biol. Chem. 277:11582-11590). Accordingly, in someembodiments, the promoter of the present disclosure comprises criticalsequences from SEQ ID NOS: 1, 2, 3, 4, or 5 that are necessary forpromoting fruit-specific expression.

Heterologous Nucleotide Sequence

In some embodiments, the promoter of the present disclosure is operablylinked to a heterologous nucleotide sequence.

In the context of the present disclosure, the term “operably linked”means that one genetic element of a genetic construct is in a functionalrelationship with another genetic element of the genetic construct. Forexample, a promoter or enhancer is operably linked to a coding sequenceif it affects the transcription of the coding sequence; or a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to facilitate translation. In some embodiments, the geneticelements being linked are contiguous. In other embodiments, the operablylinked genetic elements are not contiguous, e.g., enhancers may not becontiguous with a coding sequence.

In genetic constructs of the present disclosure, the heterologousnucleotide sequence encodes a product of interest. As used herein, a“heterologous nucleotide sequence” refers to a DNA or RNA sequence thatis from a different origin than the nucleotide sequence of the promoter.Thus, a nucleotide sequence that has been isolated from an organismdifferent from that of the promoter is considered a heterologousnucleotide sequence with respect to the promoter; a nucleotide sequencethat has been isolated from a gene that is different from that of thepromoter is also considered a heterologous nucleotide sequence withrespect to the promoter. In some embodiments, the heterologousnucleotide sequence encodes an RNA molecule. In some embodiments, theheterologous nucleotide sequence encodes a polypeptide. The encodingheterologous nucleotide sequence may be any type of nucleotide sequence,including but not limited to, complementary DNA (cDNA), genomic DNA(gDNA), nuclear DNA, organellar DNA, mitochondrial DNA, chloroplast DNA,plastid DNA, plasmid DNA, viral DNA, isolated DNA, purified DNA, andsynthetic DNA.

As used herein, the term “product of interest” refers to any biologicalproduct resulting from expression of a nucleotide sequence. For example,the product of interest may be a transcriptional product of theheterologous nucleotide sequence of the genetic construct (e.g., mRNA,tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA orany other type of RNA), or it may be a polypeptide produced bytranslation of the mRNA transcribed from the heterologous nucleotidesequence of the genetic construct.

Polypeptide

In certain embodiments, the product of interest encoded by theheterologous nucleotide sequence is a polypeptide. As used herein, theterms “polypeptide” and “protein” are used interchangeably to refer toan amino acid sequence that includes a plurality of consecutivepolymerized amino acid residues. The product of interest may includepolypeptides as direct products from translation, or it may includepolypeptides modified by, for example, methylation, acetylation,phosphorylation, ubiquitination, and glycosylation.

In one embodiment, the product of interest is a MYB transcriptionfactor. The term “MYB transcription factor” or “MYB protein” is wellunderstood by those skilled in the art to refer to a large class oftranscription factors characterized by a structurally conserved MYBdomain which is a DNA-binding domain that contains single or multipleimperfect repeat sequences. For the purpose of this disclosure, theterms “MYB”, “MYB-like” and “MYB-related” are interchangeable. Forexample, “MYB domain” may be used interchangeably with “MYB-likedomain”, and “MYB transcription factor” may be used interchangeably with“MYB-related transcription factor” or “MYB-related protein”. In plants,MYB transcription factors are involved in various processes of plantgrowth and development, including regulation of secondary metabolism aswell as response to biotic and abiotic stresses. In particular, specificMYB transcription factors have been suggested as a major determinant ofanthocyanin activation and accumulation in plants (Du et al.,Biochemistry (2009)74:1-11, Dubos et al., Plant J. (2008)55:940-953).

In another embodiment, the product of interest is a phytoene synthase(PSY). Phytoene synthase is an enzyme involved in the biosynthesis ofcarotenoids. Carotenoid biosynthesis is initiated by phytoene synthase,which catalyzes a tail-to-tail condensation of geranylgeranylpyrophosphate (GGPP) to form phytoene, which after successivedesaturation reactions is converted into lycopene. Two main types of PSYexist in plants, PSY1 and PSY2; the former being more responsible forcarotenoid synthesis in fruit ripening, whereas the latter beingpredominantly responsible for carotenoid synthesis inchloroplast-containing tissues. Manipulation of PSY expression in manyplants has been shown to dramatically enhance formation of carotenoidsand their metabolic intermediates, including lycopene (Liu et al. (2004)Proc. Natl. Acad. Sci. 101:9897-9902, Kolotilin et al. (2007) PlantPhysiol. 145:389-401, Galpaz et al. (2008) Plant J. 53:717-730).

In yet another embodiment, the product of interest is a lycopene cyclase(LCY). Lycopene cyclase is an enzyme involved in cyclization oflycopene, which is an important branching step in carotenoidbiosynthesis. In plants, there are two types of LCY, lycopene β-cyclase(β-LCY) and lycopene ε-cyclase (ε-LCY), both of which are involved inthe conversion of lycopene into carotenoids. Because LCY is involved inbreaking down of lycopene, reducing expression of LCY has been suggestedto increase the accumulation of lycopene. For a more detaileddescription of LCY, see Cunningham, et al. (1996) Plant Cell8(9):1613-1626.

In still another embodiment, the product of interest is a DXP synthase(DXS). 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) is an enzymethat catalyzes the first biosynthetic step of the2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. In plants, the MEPpathway is involved in the synthesis of the common precursors to theplastidic isoprenoids, isopentenyl diphosphate and dimethylallyldiphosphate, in plastids. DXS is recognized as limiting this pathway andis a potential target for manipulation to increase various isoprenoidssuch as carotenoids. For a more detailed description of DXS, see Lang etal. (1998) Proc. Natl. Acad. Sci. 95:2100-2104.

In other embodiments, the product of interest of the present disclosuremay be a polypeptide useful in genome editing. As used herein, the term“genome editing” or “gene editing” refers to the process of altering thetarget genomic DNA sequence by inserting, replacing, or removing one ormore nucleotides. Genome editing may be accomplished by using nucleases,which create specific double-strand breaks (DSBs) at desired locationsin the genome, and harness the cell's endogenous mechanisms to repairthe induced break by homology-directed repair (HDR) (e.g., homologousrecombination) or by nonhomologous end joining (NHEJ). Any suitablenuclease may be introduced into a cell to induce genome editing of atarget DNA sequence including, but not limited to, CRISPR-associatedprotein (Cas, e.g., Cas9) nucleases, zinc finger nucleases (ZFNs, e.g.Fokl), transcription activator-like effector nucleases (TALENs, e.g.,TALEs), meganucleases, and variants thereof (Shukla et al. (2009) Nature459: 437-441; Townsend et al (2009) Nature 459: 442-445). In someembodiments, the product of interest of the present disclosure is Cas9.

RNAs

In certain embodiments, the product of interest encoded by theheterologous nucleotide sequence of the present disclosure is an RNAmolecule. The RNA molecule may be a coding RNA, such as messenger RNA(mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA); alternatively, theRNA molecule may be one that is capable of regulating gene expression,such as a small RNA. The product of interest of the present disclosuremay also be an RNA molecule that has been modified, by processes such ascapping, polyadenylation, methylation, and editing.

As used herein, the term “small RNA” refers to several classes ofnon-coding ribonucleic acid (ncRNA). Small RNA molecules usually includeabout 20 to 30 nucleotides. The small RNA sequences may be derived fromlonger precursors. The precursors form structures that fold back on eachother in self-complementary regions, which are then processed by thenuclease Dicer in animals or DCL1 in plants. Many types of small RNAexist either naturally or produced artificially, including microRNA(miRNA), short interfering RNA (siRNA), antisense RNA, short hairpin RNA(shRNA), and small nucleolar RNA (snoRNA). Small RNA sequences do notdirectly code for a protein, and differ in function from other RNAs inthat small RNA sequences are only transcribed and not translated.Certain types of small RNA, such as microRNA and siRNA, are important inthe process RNA interference (RNAi). RNAi is a process of geneticregulation in which a target gene that would otherwise normally expressis suppressed from expression due to interference of small RNAs throughpost-transcriptional degradation or inhibition of translation. Fordetailed description of RNAi techniques, see, e.g., U.S. Pat. Nos.5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; and 6,777,588.

Accordingly, the product of interest of the present disclosure that isan RNA molecule may silence expression of a native DNA sequence within aplant's tissues to achieve a desired phenotype, for example, increasedlycopene content in a fruit. In such case, gene silencing may beaccomplished, for example, by transforming into a plant the geneticconstruct disclosed herein containing a fruit-specific promoter operablylinked to a heterologous nucleotide sequence, which encodes an siRNAmolecule that binds to and cleavages transcripts of lycopene cyclase(LCY), whereby silencing the gene expression thereof. Because LCY isresponsible for breaking down lycopene, the resulting transgenic plantwith fruit-specific silencing of LCY expression would therefore haveincreased accumulation of lycopene in the fruit.

In addition, the product of interest of the present disclosure may alsobe an RNA molecule that is useful in genome editing. Examples of suchRNA molecules include, but are not limited to, CRISPR RNA (crRNA),trans-activating crRNA (tracrRNA), guide RNA (gRNA), and single guideRNA (sgRNA). In some embodiments, the product of interest of the presentdisclosure is a single guide RNA.

Metabolic Pathways

In certain embodiments, the product of interest encoded by theheterologous nucleotide sequence of the present disclosure is involvedin a metabolic pathway. The term “metabolic pathway” refers to theseries of linked biochemical reactions involved in the synthesis,conversion and breakdown of a compound in an organism. Geneticengineering of metabolic pathways and pathway components is also knownas “metabolic engineering”. A metabolic pathway can be part of eitherprimary or secondary metabolism.

Primary metabolism refers to the sum of metabolic activities that arecommon to most, if not all, living cells and are necessary for basalgrowth and maintenance of the cells. A metabolic pathway in primarymetabolism is known as a “primary metabolic pathway”. In someembodiments, the product of interest is involved in a primary metabolicpathway. In the context of genetic engineering in fruit crops,modifications in primary metabolic pathways may lead to changes insugar, protein and lipid content in a plant, for example, fruit withimproved flavor and nutrition profile.

Fatty acids are the most abundant form of reduced carbon chainsavailable from nature and have diverse uses ranging from food toindustrial feedstocks. In recent years, long chain polyunsaturatedomega-3 fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA), anddocosahexaenoic acid (DHA) have received considerable attention fortheir health-promoting benefits. However, EPA and DHA are typicallysourced from marine fish only. Reconstitution of the omega-3 LC-PUFAbiosynthetic pathway in plants by producing transgenic plants engineeredto accumulate omega-3 LC-PUFA would be advantageous. Accordingly, insome embodiments, the product of interest of the present disclosure isin a fatty acid metabolic pathway. In some further embodiments, theproduct of interest of the present disclosure is in the LC-PUFAbiosynthetic pathway. Fruit-specific expression of such a product ofinterest may result in increased levels of omega-3 LC-PUFAs, includingEPA and DHA, to levels similar to those found in fish oil in the fruit.

Secondary metabolism refers to the biological pathways that are notabsolutely required for the survival of the organism. Compared toprimary metabolism which is more conserved throughout a wide variety oftaxa, secondary metabolism is more species-specific or organ-specific.Thus, secondary metabolism is also sometimes known as “specializedmetabolism”. A metabolic pathway in secondary metabolism is referred toas a “secondary metabolic pathway”. Compounds such as substrates,intermediates and products of secondary metabolic pathways areaccordingly referred to as “secondary metabolites”. Many of thesecondary metabolites, such as anthocyanins, lycopene, and tocopherolshave been shown to be powerful antioxidants that could be incorporatedinto human diet for potential health benefits. One way of accomplishingthis objective is to genetically engineer plants to have properaccumulation of these beneficial secondary metabolites to a level thatis health-promoting. Thus, the genetic constructs of the presentdisclosure may be used to specifically increase the contents of thesesecondary metabolites in fruit for human consumption.

Accordingly, in some embodiments, the product of interest is involved ina flavonoid metabolic pathway. Flavonoids are among thebest-characterized plant secondary metabolites in terms of chemistry,coloration mechanism, biochemistry, genetics and molecular biology.Flavonoids, with a basic structure of C6-C3C-6, are widely distributedamong land plants. Flavonoids in plants are mainly classified into sixmajor subgroups: chalcones, flavones, flavonols, flavandiols,anthocyanins, and proanthocyanidins or condensed tannins. Modificationof flavonoids with hydroxyl, methyl, glycosyl and acyl groups results inseveral thousand structures. The flavonoid biosynthetic pathway has beenwell studied among higher plants. Flavonoids are synthesized in thecytosol. It has been proposed that the biosynthetic enzymes form asuper-molecular complex (metabolon) via protein—protein interaction andare anchored in the endoplasmic reticulum (ER) membrane. Thebiosynthetic enzymes belong to various enzyme families, such as2-oxoglutarate-dependent dioxygenases (OGD), cytochromes P450 (P450) andglucosyltransferases (GT), which suggests that plants recruited theseenzymes from pre-existing metabolic pathways. Accordingly, the productof interest of the present disclosure may be an enzyme in the flavonoidmetabolic pathway or a regulator thereof. Examples of such product ofinterest may include, without limitation, glucosyltransferases (GT),acyltransferases (AT) and methyltransferases (MT), CHS, chalconesynthase; THC2′GT, UDP-glucose:tetrahydroxychalcone 2′GT; CHI, chalconeisomerase; THC4′GT, UDP-glucose:tetrahydroxychalcone 4′GT; AS,aureusidin synthase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid3′-hydroxylase; F3′5′H, flavonoid 3′,5′-hydroxylase; DFR,dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; FNS, flavonesynthase; FLS, flavonol synthase, and R2R3 Myb transcriptional factor,basic helix-loop-helix (bHLH) transcriptional factor, and WD40-typetranscriptional factor. For a more detailed description of the flavonoidmetabolic pathway, see Winkel-Shirley (2001) Plant Physiol.126(2):485-493.

In some embodiments, the product of interest is involved in theanthocyanin metabolic pathway. Anthocyanins form a large subclass offlavonoids conferring different colors typically red, purple, or blue infruits and flowers. The structural genes involved in the anthocyaninbiosynthetic pathway of plants include chalcone synthase, chalconeisomerase, flavanone 3-hydroxylase, flavonoid 3,5-hydroxylase,dihydroflavonol 4-reductase, anthocyanidin synthase, leucoanthocyanidindioxygenase and UDP-glucose: flavonoid 3-O-glucosyltransferase. Thesegenes are well characterized in model plants as well as fruit speciesincluding grape, apple and litchi (Litchi chinensis). In addition, anumber of studies have demonstrated that anthocyanin accumulation islargely regulated by MYB transcriptional factors, which manipulate theexpression of the structural genes in the anthocyanin biosyntheticpathway (Boss et al., 1996 Plant Physiol. 111:1059-1066; Niu et al.,2010 Planta 231:887-899; Petroni et al., 2011 Plant Sci. 181:219-22).Thus, in some embodiments, the product of interest of the presentdisclosure is an enzyme in the anthocyanin metabolic pathway. In someother embodiments, the product of interest of the present disclosure isa transcription factor regulating the anthocyanin metabolic pathway. Insome particular embodiments, the product of interest of the presentdisclosure is a MYB transcription factor regulating the anthocyaninmetabolic pathway.

In yet some other embodiments, the product of interest is involved in atocopherol metabolic pathway. Tocopherols are a class of lipophilicantioxidants, and together with tocotrienols belong to the vitamin Efamily. The four forms of tocopherols (α-, β-, γ- and δ-tocopherols)consist of a polar chromanol ring and lipophilic prenyl chain withdifferences in the position and number of methyl groups. Thebiosynthesis of tocopherols takes place mainly in plastids of higherplants from precursors derived from two metabolic pathways: homogentisicacid, an intermediate of degradation of aromatic amino acids, andphytyldiphosphate, which arises from methylerythritol phosphate pathway.The regulation of tocopherol biosynthesis in photosynthetic organismsoccurs, at least partially, at the level of key enzymes includingp-hydroxyphenylpyruvate dioxygenase (HPPD), homogentisatephytyltransferase (HPT), tocopherol cyclase (TC), andmethyltransferases. Accordingly, in some embodiments, the product ofinterest of the present disclosure is an enzyme in a tocopherolmetabolic pathway. In certain other embodiments, the product of interestof the present disclosure is a regulator of a tocopherol metabolicpathway that is selected from p-hydroxyphenylpyruvate dioxygenase(HPPD), homogentisate phytyltransferase (HPT), tocopherol cyclase (TC),and methyltransferase.

In still other embodiments, the product of interest is involved in acarotenoid metabolic pathway. Carotenoids are a diverse group ofisoprenoid pigments widely distributed in nature. The vivid yellow,orange, and red colors of many fruits are attributed to the accumulationof carotenoids. This physical property of carotenoids is due to apolyene chain with a number of conjugated double bonds that functions asa chromophore. Carotenoids are synthesized by all photosyntheticorganisms including plants, as well as some non-photosynthetic bacteriaand fungi. Plant carotenoids are tetraterpenes derived from the40-carbon isoprenoid phytoene. In plants, carotenoids are synthesized inall types of differentiated plastids but accumulate in high levels inthe chloroplasts of green tissues and the chromoplasts of roots, fruits,and flower petals. Accordingly, the genetic constructs of the presentdisclosure may be used to genetically engineer a carotenoid metabolicpathway by containing a heterologous nucleotide sequence encoding anenzyme in the carotenoid metabolic pathway, or a regulator regulatingthe carotenoid metabolic pathway. Examples of the enzyme and regulatorinclude, but are not limited to, the three upstream enzymes belonging tothe methylerthritol-4-phosphate (MEP) pathway that providescarotenogenesis building blocks: deoxy-d-xylulose 5-phosphate (DXP)synthase (DXS), DXP reductoisomerase (DXR) and geranylgeranyldiphosphate synthase (GGPPS); the carotenogenesis enzymes: phytoenesynthase (PSY); phytoene desaturase (PDS); ζ-carotene isomerase (Z-ISO);ζ-carotene desaturase (ZDS); carotene isomerase (CRTISO); lycopeneε-cyclase (ε-LCY); lycopene β-cyclase (β-LCY); β-carotene hydroxylase(β-OHase) carotenoid cleavage dioxygenases (CCDs); 9-cis-epoxycarotenoiddioxygenases (NCEDs); and the transcription factors regulatingcarotenogenesis: RIN, TAGL1, AP2a, ERF6, DET1, APRR2-Like, SGR andBZR1-1D. For a more detailed description of the carotenoid metabolicpathway, see Schmidt-Dannert et al. (2000) Nature Biotechnology18(7):750-754.

In some embodiments, the product of interest is involved in the lycopenemetabolic pathway. Lycopene, which derives its name from the neo-LatinLycopersicum—the tomato species, is a bright red carotene and carotenoidpigment and phytochemical found in tomatoes and other red fruits andvegetables, such as red carrots, watermelons, gac, and papayas. Lycopeneis a key intermediate in the biosynthesis of many carotenoids, thus thelycopene metabolic pathway overlaps with that of carotenoids. Generally,synthesis of lycopene begins with mevalonic acid, which is convertedinto dimethylallyl pyrophosphate. Dimethylallyl pyrophosphate is thencondensed with three molecules of isopentenyl pyrophosphate (an isomerof dimethylallyl pyrophosphate), to give the twenty-carbongeranylgeranyl pyrophosphate. Two molecules of this product are thencondensed in a tail-to-tail configuration to give the forty-carbonphytoene, the first committed step in carotenoid biosynthesis. Throughseveral desaturation steps, phytoene is converted into lycopene, whichconcludes the biosynthesis of lycopene. Breakdown of lycopene involvesthe two terminal isoprene groups of lycopene being cyclized by lycopenecyclase (LCY) to produce alpha- and beta-carotene, which can then betransformed into lutein and xanthophylls, respectively. Accordingly, thegenetic constructs of the present disclosure may be used to geneticallyengineer the lycopene metabolic pathway, by containing a heterologousnucleotide sequence encoding an enzyme in the lycopene metabolic pathwayor a regulator thereof. Examples of the enzyme and regulator include,but are not limited to, the three upstream enzymes belonging to themethylerthritol-4-phosphate (MEP) pathway that provides building blocksfor lycopene biosynthesis: deoxy-d-xylulose 5-phosphate (DXP) synthase(DXS), DXP reductoisomerase (DXR) and geranylgeranyl diphosphatesynthase (GGPPS); the lycopene biosynthesis enzymes: phytoene synthase(PSY); phytoene desaturase (PDS); ζ-carotene isomerase (Z-ISO);ζ-carotene desaturase (ZDS); carotene isomerase (CRTISO); the lycopenedegradation enzymes: lycopene ε-cyclase (ε-LCY); lycopene β-cyclase(β-LCY); β-carotene hydroxylase (β-OHase) carotenoid cleavagedioxygenases (CCDs); 9-cis-epoxycarotenoid dioxygenases (NCEDs); and thetranscription factors regulating lycopene metabolic pathway: RIN, TAGL1,AP2a, ERF6, DET1, APRR2-Like, SGR and BZR1-1D. For a more detaileddescription of the lycopene metabolic pathway, see Schmidt-Dannert etal. (2000) Nature Biotechnology 18(7):750-754.

In yet other embodiments, the product of interest is involved in thebetalain metabolic pathway. Betalains are a class of red and yellowindole-derived pigments found in plants of the Caryophyllales and somehigher order fungi. Originally found from red beet (Beta vulgaris),betalains are widely used as a natural colorant. The advantage ofbetalain color is that the color does not depend on the pH and is morestable than that from certain other pigments such as anthocyanins.Betalains are classified into red (crimson) betacyanins and yellowbetaxanthins. They are immonium conjugates of betalamic acid withcyclo-dihydroxyphenylalanine (cDOPA) glucoside and amino acids oramines, respectively. Only betacyanins are modified by glycosyl or acylmoieties. More than 50 molecular species of betacyanins and severalbetaxanthins have been isolated and identified, and novel betalainmolecules are being reported in accordance with the progress indevelopment of analytical equipment. The biosynthetic pathways ofbetalains and the enzymes and genes involved in the pathway aregenerally less well understood than those of flavonoids and carotenoids.More recently, betalains have been recognized as powerful dietaryantioxidants (Kanner et al. (2001) J Agric Food Chem 9(11):5178-5185).Thus, expression of betalains in fruits would be desirable. Accordingly,the genetic constructs of the present disclosure may be used togenetically engineer the betalain metabolic pathway, by containing aheterologous nucleotide sequence encoding an enzyme in the betalainmetabolic pathway or a regulator thereof. Examples of such product ofinterest may include, without limitation, tyrosine hydroxylase,dihydroxyphenylalanine (DOPA) dioxygenase, DOPA oxidase, DOPA4,5-dioxygenase (DOD), cDOPA 5-O-GT (cDOPA5GT), and glucosyltransferase.For a more detailed description of the betalain metabolic pathway, seePolturak et al. (2016) New Phytologist 210(1):269-283.

In some embodiments, the product of interest of the present disclosuremay be involved in a combination of the metabolic pathways describedherein.

Other Construct Elements

Genetic constructs of the present disclosure, in addition to thepromoter and the heterologous nucleotide sequence, may includeadditional construct elements that may be helpful in expressing aproduct of interest in plants. Examples of such additional constructelements include, without limitation, enhancers, silencers, promoters,5′ untranslated regions (5′ UTRs), open reading frames (ORFs), exons,introns, protein-coding regions, functional RNA-coding regions, 3′untranslated regions (3′ UTRs), terminators, transit peptides andlocalization signals, and fragments thereof. Genetic constructsincorporating these additional construct elements may be assembled usingmethods known in the art, including Sambrook et al., Molecular Cloning:A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 and Ausubelet al., Current Protocols in Molecular Biology, Greene Publishing, 1987.

In some embodiments, the genetic construct further contains an intron.As used herein, the term “intron” refers to any nucleic acid sequencecomprised in a gene that is transcribed but not translated. Intronsinclude untranslated nucleotide sequence within an expressed sequence ofDNA, as well as the corresponding sequence in RNA molecules transcribedtherefrom. A construct described herein can also contain sequences thatenhance translation and/or mRNA stability such as introns. The intron onthe genetic construct may be located within a genetic element or outsidea genetic element. In some embodiments, the intron is located in thepromoter of the genetic construct. The intron may be used in combinationwith the promoter to enhance expression, such as increased mRNAstability and enhanced translation efficiency.

In some embodiments, the genetic construct further contains aterminator. The term “terminator” encompasses a control sequence whichis a DNA sequence at the end of a transcriptional unit which signals 3′processing and polyadenylation of a primary transcript and terminationof transcription. The terminator can be derived from the natural gene,from a variety of other plant genes, or from T-DNA. The terminator to beadded may be derived from, for example, the nopaline synthase (NOS), theoctopine synthase (OCS) genes, and the 35S terminator of cauliflowermosaic virus (CaMV).

In some embodiments, the genetic construct further contains a selectablemarker. “Selectable marker”, “selectable marker gene” or “reporter gene”includes any gene that confers a phenotype on a cell in which it isexpressed to facilitate the identification and/or selection of cellsthat are transfected or transformed with a nucleic acid construct of theinvention. These marker genes enable the identification of a successfultransfer of the nucleic acid molecules via a series of differentprinciples. Suitable markers may be selected from markers that conferantibiotic or herbicide resistance, that introduce a new metabolic traitor that allow visual selection. Examples of selectable marker genesinclude genes conferring resistance to antibiotics (such as nptll thatphosphorylates neomycin and kanamycin, or hpt, phosphorylatinghygromycin, or genes conferring resistance to, for example, bleomycin,streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin,geneticin (G418), spectinomycin or blasticidin), to herbicides (forexample bar which provides resistance to bialaphos and glufosinate; aroAor gox providing resistance against glyphosate, or the genes conferringresistance to, for example, imidazolinone, phosphinothricin orsulfonylurea), or genes that provide a metabolic trait (such as manAthat allows plants to use mannose as sole carbon source or xyloseisomerase for the utilization of xylose, or antinutritive markers suchas the resistance to 2-deoxyglucose). Expression of visual marker genesresults in the formation of color (for example β-glucuronidase, GUS orβ-galactosidase with its colored substrates, for example X-Gal),luminescence (such as the luciferin/luciferase system) or fluorescence(green fluorescent protein, GFP, and derivatives thereof).

Expression Vectors

In one aspect, an expression vector containing a genetic construct ofthe present disclosure is provided. As used herein, the term “vector”refers to a nucleic acid molecule capable of transporting anothernucleic acid to which it has been linked. Examples of vector include,but are not limited to, plasmid, cosmid, bacteriophage, bacterialartificial chromosome (BAC), yeast artificial chromosome (YAC), or virusthat carries exogenous DNA into a cell. One type of vector is a“plasmid”, which refers to a circular double stranded DNA loop intowhich additional DNA segments can be ligated. As used herein, “plasmid”and “vector” may be used interchangeably as the plasmid is the mostcommonly used form of vector. A vector may be a binary vector or a T-DNAthat comprises the left border and the right border and may include agene of interest in between. The term “expression vector” as used hereinmeans a vector capable of directing expression of a particularnucleotide sequence in an appropriate host cell. Thus, a vectorcontaining an expression construct is considered an expression vector.

Methods for producing and using expression vectors are well known in theart and are described generally in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987, and Ausubelet al., Current Protocols in Molecular Biology, Greene Publishing, 1987.

Transgenic Plants of the Disclosure

In other aspects, transgenic plants and transgenic plant partscontaining a genetic construct of the present disclosure are provided.

As used herein, a “transgenic plant” refers to a plant that hasincorporated a heterologous or exogenous nucleotide sequence, i.e., anucleotide sequence that is not present in the native (non-transgenic or“untransformed”) plant or plant cell. “Transgenic” is used herein toinclude any cell, cell line, callus, tissue, plant part or plant, thegenotype of which has been altered by the presence of heterologousnucleotide sequence including those transgenics initially so altered aswell as those created by sexual crosses or asexual propagation from theinitial transgenic plant. The term “transgenic” as used herein does notencompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition, or spontaneous mutation.

Plant Transformation

Improvement of plant varieties through genetic transformation has becomeincreasingly important for modern plant breeding. Genes of potentialcommercial interest, such as genes conferring to a plant trait ofdisease resistance, insect resistance or improved quality, may beincorporated into crop species through various gene transfertechnologies. The development of an efficient transformation system isnecessary for the analysis of gene expression in plants. Therequirements for such a system include a proper target plant tissue thatwill allow efficient plant regeneration, a gene delivery vehicle thatdelivers foreign DNA efficiently into the target plant cells, and aneffective method for selecting transformed cells. In genetictransformation of dicotyledonous species, for example, transformationsystems utilizing the bacterium Agrobacterium tumefaciens have beenfrequently used as vehicles for gene delivery. The preferred targettissues for Agrobacterium-mediated transformation presently includecotyledons, leaf tissues, and hypocotyls. High velocity microprojectilebombardment offers an alternative method for gene delivery into plants.

As used herein, the term “transformation” and “transforming” a plantcell encompasses all techniques by which a nucleic acid molecule may beintroduced into such a cell. Examples include, but are not limited to:transfection with viral vectors; transformation with plasmid vectors;electroporation; microinjection; Agrobacterium-mediated transfer; directDNA uptake; Whiskers-mediated transformation; and microprojectilebombardment. These techniques may be used for both stable transformationand transient transformation of a plant cell. The term “stabletransformation” refers to the introduction of a nucleic acid fragmentinto a genome of a host organism resulting in genetically stableinheritance over two or more generations. Once stably transformed, thenucleic acid fragment is stably integrated in the genome of the hostorganism and any subsequent generation. Host organisms containing thetransformed nucleic acid fragments are referred to as “transgenic”organisms. “Transient transformation” refers to the introduction of anucleic acid fragment into the nucleus, or DNA-containing organelle, ofa host organism resulting in gene expression without genetically stableinheritance.

Methods for transforming plant cells, plants and portions thereof aredescribed in Draper et al., 1988, Plant Genetic Transformation and GeneExpression. A Laboratory Manual, Blackwell Sci. Pub. Oxford, p. 365;Potrykus and Spangenburg, 1995, Gene Transfer to Plants.Springer-Verlag, Berlin; and Gelvin et al., 1993, Plant Molecular Biol.Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants,including transformation techniques, is provided in Galun and Breiman,1997, Transgenic Plants. Imperial College Press, London.

The following are representative publications disclosing genetictransformation protocols that may be used to genetically transform thefollowing plant species: citrus (Pena et al., 1995, Plant Sci. 104,183); Prunus (Ramesh et al., 2006, Plant Cell Rep. 25(8):821-8; Song andSink 2005, Plant Cell Rep. 2006; 25(2):117-23; Gonzalez Padilla et al.,2003, Plant Cell Rep. 22(1):38-45); apple (Yao et al., 1995, Plant CellReports 14, 407-412); tomato (U.S. Pat. No. 5,159,135); banana (U.S.Pat. No. 5,792,935); pineapple (U.S. Pat. No. 5,952,543); strawberry(Oosumi et al., 2006, Planta 223(6):1219-30; Folta et al., 2006, Planta.2006 Apr. 14; PMID: 16614818); Rubus (Graham et al., 1995, Methods Mol.Biol. 1995; 44:129-33). Transformation of other species is alsocontemplated by the disclosure. Suitable methods and protocols fortransformation of other species are available in the scientificliterature and known to those of skill in the art.

As used herein, a transgenic “event” or “line” is produced bytransformation of plant cells with a genetic construct containing aheterologous nucleotide sequence, regeneration of a population of plantsresulting from the insertion of the heterologous nucleotide sequenceinto the genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. The term“event” refers to the original transformant and progeny of thetransformant, where the genetic construct is inserted in a particulargenome location. Multiple transgenic events or lines may be producedfrom one transformation process. Different transgenic events or linesmay possess different characteristics (e.g. transgene expression,desired phenotypes), depending on the copy of the genetic constructsbeing inserted into the plant genome and the location of the geneticconstruct being inserted into the plant genome.

Transgenic Plant Parts

In some embodiments, the present disclosure relates to a plant part ofthe transgenic plant, where the plant part contains a genetic constructsof the present disclosure.

As used here in, a “plant part” refers to any part of a plant, includingcells, tissues and organs. Examples of plant parts include, but are notlimited to, pollen, ovules, leaves, embryos, roots, root tips, anthers,flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts,and calli. In some embodiments, the plant part of the present disclosureis a stem, a branch, a root, a leaf, a flower, a fruit, a seed, acutting, a bud, a cell, or a portion thereof.

A plant part may be in planta (i.e., in a non-laboratory environment),or it may be in culture (e.g., cell culture, tissue culture and organculture). Plant parts include harvestable plant parts, as well as plantparts useful for propagation of progeny plants. A “harvestable part” isa plant part that may be collected for consumption and/or further use,including fruits, seeds, flowers, leaves, seeds, roots, etc. For citrusplants, the harvestable part is typically a fruit.

As used herein, the terms “propagation” and “reproduction” are usedinterchangeably to refer to the process of a progeny plant beinggenerated from a plant part of a parent plant. There are two main typesof propagation in plants: sexual propagation and asexual propagation.The term “sexual propagation” refers to generating a new plant from aseed. The term “asexual propagation”, “vegetative propagation” or“clonal propagation” refers to generating a new plant from a part of aplant of a parent plant that is not a seed. A citrus plant may bepropagated by sexual propagation or asexual propagation. The term“propagation material” or “propagating material” refers to a plant partthat is used to propagate plants. For sexual propagation, thepropagating material is a seed. For asexual propagation, the propagatingmaterial may be any non-seed plant part that is capable of regeneratinginto a new plant. For a more detailed description of plant propagation,see Hartmann and Kester (1975) Plant propagation: principles andpractices (No. SB119 H3 1975).

Transgenic Detection

After a genetic construct is transformed into a plant, a plant part, orplant cell, testing may take place to confirm that the transformationhas occurred and to assess the quality of the transformation. Forexample, when selecting among multiple transgenic events or lines thathad been transformed with the same construct, the event or line selectedshould ideally have the intact target sequences of interest withoutrearrangements, insertions, deletions, or extraneous flanking sequences.As described below, methods and techniques of detecting the geneticconstruct of the present disclosure contained in a transgenic plant ortransgenic plant part are known to those of skill in the art.

Presence

Methods for detecting the presence of a genetic construct in a plant, aplant part, or plant cell include traditional methods such as thepolymerase chain reaction (PCR) and DNA hybridization using nucleotideprobes (e.g. Southern blot; Southern (1975) J. Mol. Biol.98(3):503-517), and the more recent methods such as thermal asymmetricinterlaced-PCR (TAIL-PCR; Liu et al (1995) The Plant J. 8(3):457-463),droplet digital PCR (ddPCR; Hindson et al (2011) Anal. Chem.83(22):8604-8610). and next generation sequencing (NGS; Varshney, et al.(2009). Trends Biotechnol. 27(9):522-530). In addition, methods fortransgenic plant event-specific DNA detection are described in U.S. Pat.Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974; 6,689,880; 6,900,014and 6,818,807.

Copy Number, Zygosity and Expression

For most plant transformation methods, integration of a geneticconstruct into the host plant genome occurs randomly, i.e., the geneticconstruct may be inserted into any location of any chromosome, and anynumber of genetic constructs may be inserted. As a result, theexpression of the integrated genetic construct may be influenced by itschromosomal position, perhaps due to chromatin structure (e.g.,heterochromatin) or the proximity of transcriptional regulation elements(e.g., enhancers) close to the integration site (Weising et al., 1988Ann. Rev. Genet 22:421-477). In addition, the first generation of plantsafter transformation (“To generation”) are normally hemizygous (e.g.,only one of the two genomes of a diploid somatic cell contains theintegrated genetic construct). It is only after sexual reproductioncould homozygous plants be obtained, i.e., in T₁ generation and above.Therefore, a large number of independent transgenic events are typicallygenerated to compensate for the aforementioned uncertainties. Theresulting collection of transgenic lines can then be screened toprioritize lead events that possess targeted transgene expression levelscoupled with low-copy integrations.

Methods for detecting the copy number of the integrated geneticconstruct as well as the zygosity of a transgenic plant may include,without limitation, quantitative PCR (qPCR; Ingham et al. (2001)Biotechniques 31(1):132-141), ddPCR (supra), and NGS (supra). Methodsfor detecting the expression of a genetic construct in a transgenicplant may include, without limitation, reverse transcription PCR(RT-PCR; Stone-Marschat et al. (1994). J. Clin. Microbiol.32(3):697-700), qPCR (Brunner et al. (2004) BMC plant biology4(1):14-14), Northern blot (Alwine et al. (1977) Proc. Natl. Acad. Sci.74(12):5350-5354) and Western blot (Burnette (1981) Anal. Biochem. 112(2):195-203).

Methods of the Disclosure

Further aspects of the present disclosure relate generally to methods ofusing the genetic constructs disclosed herein.

Method for Modifying a Fruit Phenotype

Genetic improvement of plants through transgenic technology enablesintroduction of a specific trait of interest into a desirable plantvariety. In conventional breeding approaches, the trait of interest isdelivered through cross-pollination, which limits the trait to be withinthe same species or a close relative due to reproductive incompatibility(i.e., biological species boundaries); whereas transgenic technologyallows transfer of a selected trait to be across the boundaries ofgenera or higher classifications. Further, in contrast to conventionalbreeding where introduction of a trait of interest often requiresmultiple generations of crossing and selection, the transfer of selectedtrait through transgenic technology is achieved in a single generation,which is especially important for breeding of fruit trees such as citrustrees and plums that have long breeding cycles.

Accordingly, in one aspect, the present disclosure provides a method ofmodifying a fruit phenotype in a plant, the method having the steps of:i) transforming a plant cell with a genetic construct of the presentdisclosure, where expression of the product of interest is associatedwith modification of the fruit phenotype; ii) regenerating a plant fromthe transformed plant cell; and iii) growing the regenerated plant toproduce fruit of the modified phenotype.

Regeneration and Growth of Transgenic Plants

The processes of transformation, regeneration, and growth are requiredto modify a fruit phenotype in a plant. Methods and techniques oftransformation, where a genetic construct of the present disclosure isdelivered to a plant cell and incorporated into the plant genome, havebeen described above in other aspects of the disclosure. Followingtransformation, steps of regeneration and growth are necessary in orderto recover a whole and fertile plant from the transformed plant cell, asdescribed below.

As used herein, the terms “regenerate”, “regenerating” and“regeneration” refer to the process of developing a plant from atransformed plant cell. This is typically accomplished through planttissue culture, which is a collection of techniques used to grow plantcells, tissues or organs under sterile conditions on a nutrient culturemedium of known composition. Plant tissue culture relies on the factthat many plant cells have the ability to regenerate a whole plant, aproperty known as “totipotency”. Single plant cells, unorganized growthsof plant cells (“calluses”), plant cells without cell walls(protoplasts), and other plant parts (e.g., leaves, stems or roots) canoften be used to generate a new plant on culture media given therequired nutrients and plant hormones. The plant part (e.g., cell,protoplast, tissue and organ) removed from a plant to be cultured isknown as an “explant”. During transformation, not all the explant'scells are transformed; typically a selectable marker is used todifferentiate transformed from untransformed cells. In some embodiments,the genetic construct of the present disclosure comprises a selectablemarker, such that the cells that have been successfully transformed withthe genetic construct would contain the selectable marker. By growingthe cells in the presence of an antibiotic or chemical that selects ormarks the cells expressing the selectable marker, it is possible toseparate transformed from untransformed cells. The transformed plantcell are then placed onto the surface of a sterile solid culture medium,which typically comprises inorganic salts, organic nutrients, vitamins,and plant hormones for plant regeneration. The composition of themedium, particularly the plant hormones and the nitrogen source (nitrateversus ammonium salts or amino acids) have profound effects on themorphology of the tissues that grow from the initial explant. Forexample, an excess of auxin will often result in a proliferation ofroots, while an excess of cytokinin may yield shoots. A balance of bothauxin and cytokinin will often produce an unorganized growth of cells,or callus, but the morphology of the outgrowth will depend on the plantspecies as well as the medium composition. As shoots emerge from aculture, they may be sliced off and treated with auxin to produce roots,and develop into a plantlet.

The subsequent step is to grow the regenerated plantlets into matureplants that are able to produce fruit of the modified phenotype.Plantlets regenerated from tissue culture are very fragile as they havebeen cultured on nutrient media under aseptic conditions and grown inhigh humidity (nearly 100%). Under those conditions, the regeneratedplantlets tend to have: i) fewer palisade cells, i.e., lessphotosynthetic capability; ii) poorer vascular connection between rootsand shoots and thus reduced water conduction; and iii) less developedcuticle or waxy layer, which results in greater water loss throughevaporation when the plantlet is transferred to a less humidenvironment. Since the regenerated plantlets are highly vulnerable toenvironmental stress, carefully controlled acclimation procedures arenecessary for their survival. As used herein, the terms “acclimation”,“acclimatization”, and “hardening-off” are used interchangeably to referto the transitional process in which a plantlet gradually adjusts to thechanges in its environment (such as a change in temperature, humidity,and/or photoperiod). The initial regenerated plants are called T₀ plantsand the seeds obtained from the T₀ plants belong to the T₁ generation.For more details regarding regeneration and growth of a transgenicplant, see Teng, et al., HortScience. 1992, 27: 9, 1030-1032, Teng, etal., HortScience. 1993, 28: 6, 669-1671, Zhang, et al., Journal ofGenetics and Breeding. 1992, 46: 3, 287-290, Webb, et al., Plant CellTissue and Organ Culture. 1994, 38: 1, 77-79, Curtis, et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Nagata, et al., Journalfor the American Society for Horticultural Science. 2000, 125: 6,669-672, and Ibrahim, et al., Plant Cell, Tissue and Organ Culture.(1992), 28(2): 139-145.

Fruit Phenotypes

As used herein, the term “phenotype” may be used interchangeably withthe term “trait”, which refers a plant characteristic that is readilyobservable or measurable and is a result of the interaction of thegenetic makeup of the plant with the environment in which it develops.

The phenotypes that impart distinctive quality in a fruit may becategorized into 1) appearance, 2) flavor, 3) texture and 4) nutritionalvalue. Appearance may be determined by physical factors including,without limitation, size, shape, wholeness, presence of defects(blemishes, bruises, spots, etc.), finish or gloss, and consistency.Flavor is typically described by aroma (e.g. odor) and taste (e.g.,sweetness and acidity). Texture may be measured by percentage of water,percentage of soluble solids, and percentage of insoluble solids. Fruitsare a major source of both “macro” nutrients such as fiber andcarbohydrates, and “micro” nutrients such as vitamin C, vitamin Bcomplex (thiamin, riboflavin, B6, niacin, folate), vitamin A, vitamin E,minerals, polyphenolics, carotenoids, and glucosinolates. In addition,parameters related to juice quality are also important fruit traits,which include, without limitation, juice yield, total soluble solids(TSS), total acidity (TA), TSS/TA ratio, pectin content, pulp content,and fiber content. Further, characteristics related generally to thegrowth and development of fruit trees are also considered importantfruit traits, which include, without limitation, fruit yield, fruitripening and senescence (e.g., post-harvest shelf life), resistance todiseases and insects, tolerance to environmental stress (e.g. drought,heat, salinity), and resistance to herbicides.

In some embodiments, the fruit phenotype is selected from the groupconsisting of size, weight, color, shape, firmness, glossiness, flavor,aroma, secondary metabolite content, peel thickness, seed number, juicequality, juice sugar content, juice acid content, juice taste, juicecolor, and juice yield.

Types of Fruit

The method disclosed herein is applicable to a number of different fruitspecies. As used herein, the term “fruit” refers to its botanicalmeaning, i.e., the seed-bearing structure in flowering plants(angiosperms) formed from the ovary after flowering. Thus, in additionalto the culinary fruits, fruits as used herein also include structuresthat are not commonly regarded fruits by culinary or common meanings,such as bean pods, corn kernels, tomatoes, and wheat grains.

Examples of culinary fruits include, but are not limited to, apples,pears, grapes, drupe fruits (e.g., peaches, apricots, nectarines, plums,pluots, cherries, greengages, apriums, and peacotums), citrus fruits(e.g., mandarin, tangelo, orange, lime, lemon, meyer lemon, clementine,grapefruit, pomelo, blood orange, and calamansi), berries (e.g.,raspberries, strawberries, blueberries, blackberries, loganberries,lingonberries, cranberries, red currants, black currants, acai, gogi,bilberries, boysenberries, huckleberries, salmonberries, and acerola),papaya, cactus fruits (e.g., pitaya, prickly pear), melons (e.g.,honeydew, cantaloupe, canary, watermelon, and galia), pumpkin, avocado,guava, cherimoya, pomegranate, banana, kiwi fruits, palm fruits,persimmon, tamarind, mangoes, pineapples.

Examples of fruits that are often considered vegetables in a culinarysense may include, but are not limited to, cucumbers, tomatoes, peppers,eggplants, pumpkin, beans, nuts and cereal grains tomatoes, cucumbers,squash, zucchinis, pumpkins, peppers, eggplant, tomatillos, okra, andavocado.

In some embodiments, the fruit of the present disclosure is selectedfrom orange (Citrus sinensis), mandarin (Citrus reticulata), lime(Citrus aurantifolia), grapefruit (Citrus paradisi), lemon (Citruslimon), pomelo (Citrus maxima), citron (Citrus medico), papeda (Citrusmicrantha), citrange (Citrus sinensis x Poncirus trifoliate), and Prunussp.

Secondary Metabolites and Nutraceuticals

“Secondary metabolites” refer to compounds such as substrates,intermediates and products of secondary metabolism. Secondarymetabolites usually do not appear to participate directly in growth anddevelopment. They are a group of chemically very diverse products thatoften have a restricted taxonomic distribution. Secondary metabolitesnormally exist as members of closely related chemical families, usuallyof a molecular weight of less than 1500 Dalton. Secondary metabolites inplants include e.g. alkaloid compounds (e.g. terpenoid indole alkaloids,tropane alkaloids, steroid alkaloids), phenolic compounds (e.g.quinines, lignans and flavonoids), terpenoid compounds (e.g.monoterpenoids, iridoids, sesquiterpenoids, diterpenoids andtriterpenoids). In addition, secondary metabolites include smallmolecules, such as substituted heterocyclic compounds which may bemonocyclic or polycyclic, fused or bridged.

In some embodiments, the fruit phenotype relates to the content ofsecondary metabolites in a fruit, including anthocyanin content,tocopherol content, fatty acid content, carotenoid content, lycopenecontent, betalain content, and flavonoid content.

Many plant secondary metabolites have value as pharmaceuticals ornutraceuticals. For example, plant phenolic compounds such asanthocyanins and flavonoids have been shown to have antioxidant andanti-cancerous properties. The antioxidant activity of these compoundsis attributed to their ability to transfer hydrogen atoms or electronsof an aromatic hydroxyl group into a free radical, generating a morestable phenoxyl radical (Duthie et al., 2003), or to their ability tochelate metal ions such as iron and copper thereby acting as scavengersof singlet oxygen and free radicals (Rice-Evans et al., 1997). Today,this group of plant compounds is of great nutraceutical interest fortheir contribution to human health. As used herein, the term“nutraceutical” refers to a food or food component considered to providemedical or health benefits, including the prevention or treatment ofdisease. In some embodiments, the fruit phenotype is increasednutraceutical content.

Method for Creating Seedless Tomato

The present disclosure is based, at least in part, on the surprisingfinding that transgenic tomato plants (Solanum lycopersicum) that havebeen transformed with genetic constructs comprising a promoter sequenceof SEQ ID NO: 4 produce fruits that are without seeds or with smallnon-viable seeds. Thus, in one aspect, the present disclosure provides amethod of creating a tomato plant with seedless fruit, the method havingthe steps of 1) transforming a tomato plant cell with a geneticconstruct of the present disclosure, where the promoter comprises thesequence of SEQ ID NO: 4, or a sequence having at least 90% identitythereto; ii) regenerating a tomato plant from the transformed tomatoplant cell; and iii) growing the regenerated tomato plant to produceseedless fruit. Methods of transforming, generating and growing atransgenic plant are well known in the art, as described above in otheraspects of the disclosure.

As used herein, the term “seedless” refers to the state of containing noviable seed. Thus, a seedless fruit may contain no seeds, or it maycontain non-viable seeds. As used herein, the term “fruit” refers to itsbotanical meaning, i.e. the seed-bearing structure in flowering plants(angiosperms) formed from the ovary after flowering. Thus, fruits asused herein include structures that are not commonly regarded fruits byculinary or common meanings, such as tomatoes, bean pods, and cerealgrains.

Seedlessness may be an important trait to meet the preference ofconsumers. Examples of plants where varieties producing seedless fruitshave been selected and popularized include watermelons, grapes,citruses, pineapples, bananas, tomatoes, and peppers. Two mainmechanisms have been suggested to be responsible for the formation ofseedless fruits: (i) parthenocarpy, where the fruit develops in theabsence of fertilization, as in cultivated pineapples, some Citruscultivars, and bananas; and (ii) stenospermy, where pollination andfertilization are required, but embryos either do not form or they abortbefore completion of seed formation, as in seedless watermelons and manyseedless grapes. In both cases, the plant must have an inherent oracquired ability to sustain fruit development in the absence of seedformation.

Without wishing to be bound by theory, it is thought that the mechanismof the seedless fruit of the present disclosure is related to genomeduplication. Without wishing to be bound by theory, it is thought thatthe promoter sequence disclosed as SEQ ID NO: 4 interacts with thetomato genome, leading to genome duplication (see Example 4 and FIG. 7),which either triggers the known parthenocarpy or stenospermy mechanism,or acts through a novel mechanism to result in formation of seedlesstomato fruits.

Accordingly, in some embodiments, a promoter comprising the sequence ofSEQ ID NO: 4 may be used to produce seedless tomato. In someembodiments, the genetic construct used to create a seedless tomato maycomprise a promoter sequence that is not completely identical to SEQ IDNO: 4, but has a certain degree of sequence identity, such as at leastabout 50%, at least about 55%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 91%, at least about 92%,at least about 93%, at least about 94%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, or at least about 99%identical to SEQ ID NO: 4.

A plant promoter typically comprises a core promoter, and often,additional regulatory elements. The core promoter is the minimalsequence that is required for directing basal level of expression,comprising a TATA box region where RNA polymerase, TATA-binding protein(TBP), and TBP-associated factors may bind to initiate transcription. Inaddition to the core promoter, further sequence elements are oftennecessary for initiating transcription that has a tissue- ordevelopmental stage-specific expression pattern. For instance, theTGTCACA motif has been found to be a cis-regulatory enhancer elementnecessary for fruit-specific expression in melon species (Yamagata etal. (2002) J. Biol. Chem. 277:11582-11590). In some embodiments, apromoter comprising critical sequences from SEQ ID NOS: 1, 2, 3, 4, or 5that are necessary for promoting fruit-specific expression may be usedto produce seedless tomato.

EXAMPLES

The present disclosure will be more fully understood by reference to thefollowing examples. It is understood that various other embodiments maybe practiced, given the general description provided above. They shouldnot, however, be construed as limiting any aspect or scope of thepresent disclosure in any way.

Example 1: Identification of Candidate Fruit-Specific Promoters

The following example describes the identification of candidatefruit-specific promoters in citrus and plum.

Materials and Methods

In Silico Identification of Candidate Fruit-Specific Promoters

Candidate citrus genes with fruit-specific or fruit-preferentialexpression patterns were identified from analysis of microarray geneexpression data at the Gene Expression Omnibus (GEO) data repository(www.ncbi.nlm.nih.gov/geo). GEO is a public functional genomics datarepository supporting MIAME-compliant data submissions, where array- andsequence-based data are accepted and tools are provided to help usersquery and download experiments and curated gene expression profiles. Thepromoter sequence for the corresponding genes was predicted and obtainedfrom the Citrus sinensis annotation project(http://citrus.hzau.edu.cn/cgi-bin/blast/blast.cgi) and plexdB.org. The‘Microplatform citrus’ program was utilized for BLAST analysis of theAffymetrix IDs. The ‘GBrowse’ program was used for predicting exon andintron sequences of the candidate genes, and the 1-2 kb sequenceupstream of the start codon was predicted to be the promoter sequence.

Sequence data for candidate plum genes with fruit-specific orfruit-preferential expression patterns were obtained from the USDA plumgenome sequence database. Briefly, leaves of the European plum (Prunusdomestica) variety ‘Improved French’ were ground in liquid nitrogen,from which genomic DNA was extracted using the EZNA™ High Performance(HP) DNA Kit (Omega Bio-Tek, Norcross, Ga.) with the addition of 2%Polyvinylpyrrolidone-40 (PVP-40) (w/v) to CPL buffer and2-mercaptoethanol. Genomic DNA quantity was assessed using the Quant-iTPicoGreen kit (Invitrogen, Carlsbad, Calif.). A total of 2 μg ofpurified DNA was provided to David H Murdock Research Institute,Kannapolis, N.C. for library construction and sequencing. A paired-endand a mate-pair library were constructed with an average insert size of375 bp and 2,950 bp, respectively. These libraries were sequenced usingan 11lumina HiSeq 2000 sequencer. A total of 194,856,870 100-bppaired-end sequence reads and 158,319,386 mate-pair sequence reads wereobtained. Sequence reads were assembled against the peach (Prunuspersica) genome version 2 (Verde et al., BMC Genomics (2017) 18:225,https://www.rosaceae.org/species/prunus_persica/genome_v2.0.a1) usingthe CLC Genomics Workbench reference assembly tool (Qiagen, Valencia,Calif.) with two modifications to the default settings: lengthfraction=0.7 and similarity fraction=0.85. The −2000 bp assembledsequence upstream of the known Prunus domestica MybA gene was predictedto be the promoter of the gene. Primers were designed based on theassembled Prunus domestica genomic sequence (with the reverse primerpositioned inside the open reading frame of MybA) to amplify across thepredicted region for isolation of the candidate plum promoter PfeMybApfrom the wild/feral plum Prunus americana.

Results

To obtain candidates for novel fruit-specific promoters with uniqueactivities, novel fruit-specific genes were identified. Genes thatshowed fruit-specific expression were selected using gene expressiondata from www.ncbi.nlm.nih.gov/geo/. The microarray databases fromseveral different citrus tissues were used to identify candidate citrusgenes for fruit-specific expression. Consequently, four citrus geneswere selected based on this analysis (candidates #1-4), which show ahigh level of expression in fruit and flower with some degree ofexpression in peel but not in leaves (FIG. 1). The target descriptionand Affymetrix ID of these four candidate citrus genes are shown inTable 1. Additionally, one candidate plum gene (candidate #5) wasidentified and included in further studies.

TABLE 1 Candidate fruit-specific citrus genes Candi- Affymetrix ProbeGenBank Target date Gene ID Set ID Accession Description #1 Cs7g10980Cit.144.1.S1_s_at, CB293157 Sepallata3 Cit.29312.1.S1_s_at MADS-boxprotein 4 #2 Cs1g02750 Cit.11241.1.S1_s_at CX049273 Aldehyde decar-boxylase, WAX2, CER1 fatty acid hydroxylase #3 Cs5g31450Cit.29634.1.S1_at CK935639 Unknown #4 Cs6g16160 Cit.12380.1.S1_atCF509979 Cl111 juice sac promoter

Candidate Fruit-Specific Genes and their Promoters

Citrus-derived candidate #1 has a target description of sepallata3MADS-box protein 4 and is hereinafter referred to as CitSEP. The 3.56-kbsequence upstream of the start codon contains the promoter plus thefirst intron fragment of the gene, hereinafter referred to as CitSEPp.Because the first intron of the Arabidopsis SEP3 ortholog has been shownto be important for floral specificity when tested by the promoter fusedto a reporter gene, it is likely that the citrus first intron isrequired as well.

Citrus-derived candidate #2 has a target description of aldehydedecarboxylase/WAX2/CER1 fatty acid hydroxylase, hereinafter referred toas CitWAX. The corresponding promoter is hereinafter referred to asCitWAXp.

Citrus-derived candidate #3 has an unknown target description, and ishereinafter referred to as CitUNK, with its promoter referred to asCitUNKp. The orientation of the gene in the genome was confirmed basedon presence of poly-A tail on an EST, despite this orientation being inconflict with the annotated C. sinensis genome sequence. Based on thebioinformatics data, promoter CitUNKp should control fruit specifictranscription although the encoded protein has an unknown function.

Citrus-derived candidate #4 has a target description of C1111 juice sacpromoter (Sorkina et al. Plant Cell Rep. (2011) 30:1627-1640). Thispromoter is hereinafter referred to as CitJuSacp.

Plum-derived candidate #5 has a promoter sequence obtained from theferal plum MybA gene (PfeMybA). The promoter is hereinafter referred toas PfeMybAp.

Conclusion

Taken together, the results indicate that novel fruit-specific promoterswere successfully identified from analysis of microarray expressiondata. Candidate fruit-specific promoters include four derived fromcitrus (Citrus sinensis: CitSEPp, CitWAXp, CitUNKp and CitJuSacp) andone from plum (Prunus americana: PfeMybAp).

Example 2: Isolation and Sequence Characterization of the Fruit-SpecificPromoters

As described in Example 1, four citrus promoters and one plum promoterwere chosen for further studies. The following example describes theisolation of nucleotide sequence, generation of genetic construct, andcomparison and annotation of the various elements of the fruit-specificpromoters.

Materials and Methods

Promoter Isolation

5 grams of young leaves were harvested from trees and frozen in liquidnitrogen. Tissue was ground to fine powder from which genomic DNA wasisolated. The Gentra Puregene DNA Purification Kit (Qiagen, Valencia,Calif.) was used following manufacturer's extraction protocol for‘Frozen Leaf Tissue’. Primers used for PCR amplification of the promotersequences are summarized in Table 2.

TABLE 2  Primers used for PCR amplification of the promoter sequencesPromoter Primer Sequence SEQ ID NO. CitSEPp CitSEP_FOR1_5′-ttttGGTACCCCTGCAGGGCCATGGG 6 KpnISbfI 61 AGAAGGTGCACATACTTTAG-3′CitSEP_REV1_ 5′-ttttCCCGGGATCGATTTTCTTCTCC 7 XmaIClaI55TTTCTTTCTTCTTCTATCAC-3′ CitSEP_INT  ttttATCGATCTCCAATAGAGGAAAGCTG 8FOR2 ClaI54 TACG-3′ CitSEP_REV10_ ttttGCGGCCGCGTTTAAACGTTGCACTT 9NotIPmeI55 CTGGTACCTCTC-3′ CitWAXp CitWAX_REVl_5′-tttCCATGGTGCACTTTGAGGTAATG 10 NcoI 61 CAACATGCAATTGCTAG-3′CitWAX_INT  5′-tttGAATTCGAGAGGAAGAGAACAAC 11 FOR2_EcoRI 59AAATTAATAAAGGCGG-3′ CitUNKp CsUNK_FOR  5′-aaaaGAATTCCCTCAATCTGCACCAC 12EcorI 58 TAAGACGAAT-3′ CsUNK_REV  5′-aaaaCCATGGTTGTCTGTGGCATTCA 13NcoI 59 CTGGAGAG-3′ CitJuSacp CitSin JuSac_5′-tttGAATTCGAGAGGAAGAGAACAAC 14 FOR2 EcoRI 52 AAATTAATAAAGGCGG-3′CitSin JuSac_ tttCCATGGTTTTTTCTATTTCATTCTTT 15 REV1 NcoI 53CAGATTTTAAGC-3′ PfeMybAp PfeMybA SbfI F61 5′-agtcCCTGCAGGGATTTTCCACCTAA16 TTGCACATCGATCCAAACG-3′ PfeMybA NcoI R59 5′-agtcCCATGGTTTTCTTTTGGGCAGC17 GTTGTATGCTTGCAGC-3′

Molecular Constructs

The fruit-specific promoters, together with control promoters (i.e. thedouble enhanced CaMV 35S promoter 35Sp, the E8 promoter E8p, and the PGpromoter PGp), were PCR amplified, digested, and cloned intopCTAGII-GUSPlus vector (deposited in GenBank at NCBI with accessionnumber MG818373) for DNA sequencing. A diagram showing the molecularconstructs is presented in FIG. 2.

DNA Sequencing

Molecular constructs that contain the fruit-specific promoters wereprocessed for DNA sequencing. 1 milliliter of E. coli liquid culture iscentrifuge-harvested with supernatant disposed. Tissue was ground tofine powder from which genomic DNA was isolated. ZR plasmidminiprep-classic (Zymo Research Corp.) was used following themanufacturer's extraction protocol.

Analysis of Promoter Elements

Analysis of putative cis-regulatory elements within the fruit-specificpromoters was performed with the Plant Promoter Analysis Navigator, thePlant Cis Acting Regulatory Element (PlantCARE) search tool, and theDatabase of Plant Cis acting Regulatory DNA Elements. Additional knowncis elements that were not included within the above websites' databaseswere queried and annotated manually. Websites of the tools used in thisstudy are listed below:

http://plantpan.mbc.nctu.edu.tw/index.phphttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/http://www.dna.affrc.go.jp/PLACE/http://133.66.216.33/ppdb/cgi-bin/index.cgihttp://element.mocklerlab.org/ http://www.mocklerlab.org/toolshttp://element.mocklerlab.org/motif_finders/new.

Results

Promoter Isolation and Sequencing

Approximately 1.0-3.0 kb of the region immediately upstream of atranslation initiation codon (ATG) was predicted to be the promoterregion in each case. The promoter sequences were PCR amplified fromsweet orange and feral plum genomic DNA, and cloned into the pCTAG2vector series. Promoter sequences based on DNA sequencing of thesevectors were used for further element characterization. Nucleotidesequences of the isolated promoters CitSEPp, CitWAXp, CitUNKp, CitJuSacpand PfeMybAp are included herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ IDNO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively. Sequences are listedbelow.

SEQ ID NO: 1; Citrus sinensis; Name: CitSEPp (SEQ ID NO: 1) 5′GCCATGGGAGAAGGTGCACATACTTTAGGCATCTGATGCAAATCAGAACTAAAAAATGaAGAGAAATCTGGATTTATATATATATACTGATCTATTCTTATCTTTGTACCTTGTTTTATTTTATTTTATTAAGTAAAATCAATCACTTGTTATCTTTATTTTTCAGACAATCCCGAGGGGTAAACCAGTGAATTTATATAGAAAACAACGGAACTACGAAGCTGTTCTGTTTCAGCTTTACACGTAATTGGCCGAAGGAAATAGTCAGGTGGGGATAATCAAAAACCTCGTTCACTTCTCATCTCGACACGTGTCAATGTCCATTTATTTAATTACCTCACCTCTCCTCTTCTAAGTCTGGGATTTCCCTTATTTATTTTTTTAAGAAAAAAAATGTCTAAGGTTCCCCCCCCCCCCCCtATGGCCTCTCCACCGTCTGATCAAAGAAATAGGGTATAATAATAACAACAATAAAAGTAAAAATAAAGGAATGCAAAGCTAAAAGCAAAATAACGCTCCATAATATTCGTTTTGTTTTACATTTATATTTTTTTTGATAcATTAAATCAtCTaGTATTTGAAAATCACATTgGACCCTGATTAATTCAaATTCGAGCtAAGTAGGACCACTAGGACGGTAAAGTTCTCTCCCGACTAATCTAAATTCGAGTCGAGTAAGATCACTAAGGCAAATTCGAGCCGAGTAAGATCACTCGGCGGCAAAATTCTCCCAACAAGTATTTaATGCAtTTGTATTCTcAAGTCTCGAATCGAAGACTTTGGTTAAGTTAGAACAACCTCATACTAGTTGActcACGCGCTTGTTaGTTTGTTATACACTTATATGATAACAATAAGAGTCAAAATGAAGTCATACaGACCtaaTAATAATAATAATAATAATAATAATAGGGCAAAAAGAAAAGGTATAGGAAAGAGATCGAAGAaGCAATAGCGGAGGCAATATAATATAATACTAGAAGTGATAGATTATAAATAGATATATGAATATATAGTGATAGAAGAAGAAAGAAAGGAGAAGAAAgacgtcGCTCTCATCATTTTCTCCAATAGAGGAAAGCTGTACGAGTTTTGCAGTAGTTCAAGgtatacgcatatgcacacagatatgttatcaccaaaacaactaaacagctacaattaaatgaaaattatgaaacacaacacaaaaagctctctctttctctctctctctctctctcttctcacttggcttagctagggggggtctatgggagattctttcttttgcttggtttcttgttttgaattccgactttggatcttgaaaccataagaaatattattttttgctgtttttgatcatcccaaagaaaaaaatattgataaagaggagaaagtattgtttctttggagactggagttgagttttttgcccttttggtgaatgtctcggcgtttttagcagcttcactgtttccctcttcttattcttgtttagatctgcaactgcaaaattcatcaaaagaagatactcacacacctcactcttacatcttttaatgtattattatcactgttatattcactctaattaatttcctctctttgtttttctttttctccctgtcttttttcttttttccatttgttttcgcctattcacactttcatttccatttttgttttcttctttttccgtttttgcttcattttttttttcttcttacttcgaaaaagttcacctgatctattaatattcaatttttccaaagcaaatcaaacctaatttcaagtagtcaccttattttttgttctttataagtaaatttactggttcttccaaatagttcaagctattttctttattttagtttaattagatctcatgaagcttaaacatacaaattctgatagagggagagcgattttttttttttttttgggtattttatttcatgctttctgcagttttcagcccaaaaaaaaaaaaaaatcatgcttaatttctgttttgatgagtctgaccaaatcaagccaaatatttaagacatttattagtgatttacccagctcaaattgtgttcttgatcaaggttagttctttctgttgtatgagagttttggttctttccagtggatcatagcgttgttctttttatggagacatctccatatctgctgctgctgctgctgctttcagagcttaagctagggtttcatcttcccaaagttacttttgattttaagcttccttctttctcacacaaacacacacatgattcagatctgaactatttatgatgaattgactattgacatgttaagactgatttaactacatctaatctttaacttctttttataatttttatcatttattatgtatgaaaaaaatagggtttttttatttgtacattcactggattagaagttaatatttatcatgttcttttctgtctttttattttattttactttattttttcttgggggttaaattcggatggcatacaatctacacaataacttctgagttgtgtggaatacaaaatggattaacaaagagattttaaggaaattggaaaaggtgattataacctagatataATTCCccctcccccccccccccccaaaaaaaaaaacactttcttacaatacttcgctagataatttgatgttttattaatttttaatgtacaatgagggaaattaaagacagcttgatttacagtccccatatgttatttaactttttaaaaaaaaattgagggacataaaaatctcaagattaaacctaaagatttaggccttttgaattgaggcctatatcttctttttctttctctcccattctaatttaaaacttatcaataattaccttgccagtgcaaacactagctagtgactgatgttcatgtccatgcatttgtggagggttaattaataatgtattccttttttcattaataaattttatgcagatggaaacataatactagaaactgaatatttatttttctatcaaattgtttcctaagaactgaaacaggctctaaagcattaaccaaaccgatcctattggggttccaaaattttcttccttcctttccagtttcacccaatatatattaatctattgtgtggtttcattcaaagtcaaaattgtttttggtataacctttcatgcaatagttttcaattatttgttctctcattgtgattgattgttcagtaataatagttaatataactatcagtgcgtgagtgcgttcatttaatttgatgtgttatataatgccttttttttttttttttttcaattcatctttcattgttgactaatatatttatgcaatttgcggagggctaatgtattccttttctttgataaccccatgcgaaaatttaattagCATSEQ ID NO: 2; Citrus sinensis; Name: CitWAXp (SEQ ID NO: 2) 5′GAGAGGAAGAGAACAACAAATTAATAAAGGCGGAGCAATGAATGCATGACGTCAAAAAATTCCTGCAGAGGTTAAGACAGAGTGCACAAGCACAGAAGCGAGCAGGTATCTGAAAACATGTATTTGATCTTTATTGGGGTAGCAAAAGCCGGTGAGAACAATAAATGGTTGTCGGTGACAATTATAAACAATTGGGCCTAGTTGCACCTGCACTGTATGCTTTTATTATTGTTTTACTTTTTACTCTAGCACAATTTTACTGAAAAATGTCTTTTGCCCTCACAATACTCTTTATTCTTTATGCTTAATTATCATATTATCATTTCTTTCATTTTTTTTTGAAAAAAAAATAATTCTTATTTAAGAATTTAAATCAACTACAATATTTGTTTAATAGGACAATAACAGTTTTATATAAATTTTTTTCACTCCTAATTTTATTTTTTTGAGATAAGATTTAAAAAAGAAACACCAATACACCATTATTATTTTTTAACTTCTTATTTTAACTCCTCTATTTTATTATGATATTTACAAGTAATTTAAAGTTAATACGTCCTCTTAATTATCAATGAGAGTGGATTTAACTTATTTTGAACTTAAATTTTGATTTAGATATTCAAACTAATTCGTATAATTGATTTAGTATATTCAAATAATTTACTTGTATAATTTTTTTTTTAAATTTAATGTATAGTAATGACTCTATATTTTTATTCATAAACCTTTATTTTTTTGATTAATTATTTTCTTAAGGAAAAAATTAAACAAATATATAAAGGAC GATTGTGTTACAGAGAGCATTTAATAAAGCACCAATGGAGAAAAGGAACACTTGTCGCAGGAGCGACTGACCCTAGCACTGCTCCTATTATTCCTTAGAAGAAGGGAGCGACTGACGCTAGCACTGCTCCTATTATTAATTGTATTTTTTTTTTTAAAAAAAAGAAAAGAACCTTAATTGCTGCTACACACTTTAATGTGATAATTAAATAATCACGTGAGAGCTGGGGGTGAGCTAGCTGTAGCTGTGACATTTTTAATTGAGGCCAACAAAATATCTCCACGTGTAACCGTAATGTTGAATACCCAATTGGGCTTCGGGAAAGAAAAATTCCCCATTGATTGATCTCTCATTTGACTTGACCGTCCTGATGATGACACGACATCTAACTTGAATCCATCATCCGAATGAACAAGAACATTATATAATTAGCACCCCTCCAGCTCTACTAGCAATTGCATGTTGCATTACCTCAAAGTGCAAACAAAGA  SEQ ID NO: 3; Citrus sinensis; Name: Cit UNKp(SEQ ID NO: 3) 5′cctcaatctgcaccactaagacgaatgacaagtgagctgaaacaataatataaaaatgtaaaactgtgaatcaattacaacaattgcatctaattagatgcacagatagactttgaaagtttgcaaagtccagccactcttggtaaactaataacggcattaattatgtttattaataacattaaaataatataagcaatatgactcataatctaaaataattttgagctaagacctttagaataaactctggtcgaatagtaattcaggattataactaattaagtaggctcaaaatttttataacagatagtataaaatattagatattattatttatactgatatttaatatatataatttaaatacattattcatttattaataattatagaaataaaatcaaaattaaaaatgaatacaaggaaatggggcaatgggtaggggatggggattccacctcgttctcgtcccttcctcgaataagaaattgagtatagacacacatgtatacatacatacatatatcctctattagaaatctggaacaactggtattattatatactattccattgaaaaaaatgagacacgaatatggagtaaatgtgagaaactaattagggaaatttggctagttttttatgataaactacttacatcagtccaaagaaacatttatgggacatacccttattctctagccatgcattgatgattcatcataaaagtgtgcatgactgaaatagtcatgtgatcggccatgtcagtatctcaaactagattaaattgcaaaacaattcatcacgtcgtatattgataatttattgtcgtataaggatttatactactgtaatgattcatatacagaaaaagaaactgttgcaattagggctgcaataatggatcgatcgaaatgacaataagacaaattatgaagtaaaggctgtttttttttttttctaaatgaaacataagctatttaattttccttttgtttttatgtaaattggacttttactattagagttggactattggccattggcactcagctaatctcttcatgaatccttttttttttgttatagttattttattttcaaaaatatcattttcttaaacgcactactctaaatattttatttaaatttttttattgttataactcaaagtagtttcgtactatatttcattttttttgcactcttattgttactgtatatacatattaaaaagtattatgagtgataaaattttcaagtgaagttttataaggataacaaagggatgccagtaactttactcttactgttatagcgattcagcccaaagtaaatgtatatatattatttatttaaaaaaataagagagagaaatttagtgggtcaaaacgcattacctcaatatcattaagcagataagttagatgagtcattaatgagacccatcaacttaaattgatagaattttgaggaagtttttgatgttcgtgaagcaatgctttttatccatttactattaacttctcgtatatgcatattagcattattaattaaaatatacatatgccaaatagtgaattgtaaagaattatttcatgaatatccaataattatttaatttcttaaaattagtgggactcagcaaccctacccaagtgatagctttaattttgtaggcacaccatccaaacatgatttctctgattattattgtttaaagagtgagaattacttacatgggttaggggtcaccacctcaacatattaaaatgatgtgtttGGCTAATAAAAACTTACTAGTTTAGTGGGTACACTGCGAAACCCACTAATTTTTTTATTTTAAATAAAGCCTACCGAATTAAATTGGATGAGTCCCGCGGCAGCACCTATCTAATCGAGTCTAATGACAAAATAGAGTAAAAT GAAGGATTAATCTGAAGTCTGCTTTACTGTTTCGGCTATAAGTAAAGGAGTAGTGACCAAGACTCTCCAGTGAATGCCACAGACAA SEQ ID NO: 4; Citrus sinensis; Name: CitJuSacp (SEQ ID NO: 4) 5′AATGATTTGCAGATGCACTATAACATGGCTAATTGTTATAAAGGGCATAAATCCACCGATCACGTGATACCTTGTACTTTTATGAAATATTCATTAATTTTTTTTCTTTATAATGTCTATCTGAAATTTATAAAGTATATCACTTTTTTACCTTTTCATTAACATCCTTAAAAGATTTAACATAATATTCACAAAATTTTTCTTCTGGATGTAAAAAGGATATTTAATCTTCTTTAAACGATAAAAAAGATTTCATCTTGCAGTTGTTGGGATTAATAATAATTACAAAACTATCTATAAAAACTCATCAAATTACGTATATAAAATCATAAAATTACCAAAAGAAGCACTGTAACTAATTTGTAGCTTATTTACAACATAAATCAAGAACTCATGCTCATATAATTCATCTTAAATGACACGTCTTTTCAACAGTAACAAACTTTAACAGAAATAAATAAAAATGATCATAGTTATCTAAAATGCATTCGAAATAATCATAAAATCATTTATGAGAAATCTTGAACATTATACTTTACTTCCATAAAAAAAAAAATAATAGTATAAAACTAGTTAAGATAATCTTGGAGTTTACAGCTTATTCCCATCAATCCAAAATAATAATACATTCTCGAAGCATTGAAAATAATAATCAATGAATACTCTTTTTATATTTAGGGATAAAATAATTATTTTTTAACATGTTGTTAACCCTCTAATGGTGCTAATTAAAAAAATAAAAACTAATAAATTTTATAAACTTCACATAAAAAGCTGTAAAATAAAAAATATTTAATATAATTTTATAAAATATAAAGTATTAGATGATAGTATAAAAGCAGTAAATATAATGGAGTTACTTCACTGTAAATTACAAATTTAATATTTATTTCTATAATTATACAGTCGTTAATAATGCTGCATCGTAAAACAGTTATAACATGATTAGATTCCAGTATGAAATATCGTCTATGTGGCTCCAATAGAGTAATGACAGCCACCCTTCCGGAGAAAAAGGCAGAGAGCGGACGATTCGAATCTGGACATCTTGTTGGCGACTGGAGTGGGGAACGTGTAACAATGTCATCAACTCGTCAAACCAAACTTTCATTAAATCAATTAATTACATGGTAGTTTTGATGCCTTAAAGTCTTTGTGGTAAGTAGGAACTACCTACCAACTCTTCCCCCATATTTTATAAGAAGAATAAGAACAGCATGCGCCAGTGTTGCTCTTCTTACTTCTGCTTAAAATCTGAAAGAATGAAATAGAAAAAA SEQ ID NO: 5; Prunus Americana; Name: PfeMybAp(SEQ ID NO: 5) 5′CCAAGCTTGATTTTCCACCTAATTGCACATCGATCCAAACGCTATCCCTCTATCCCTCCAATTAAATTATGTAGCTTCCTCTTGTTCTTCACGGGCTAAAATTCTATGTTTGCTATAGTGTAGTTTCCACCAATGCCCCGTTTAAACTACAAATCAATCGGTCGTGTTTGAGCTTTTTGAATATTATCTTTTTACTTCATGTAAATTATTGTTTTCCTCTTTCAACTTAATCATATATCGTCCAATATTATTCTTGTTGAAGTTTTGTCCCTTTTTTTAACTCTAAAGCTGAATTCCTATAAAGGCTTGTAGTTTAAGTGGTTAAGAACACTTACTCATACACAAGTCCTTGCTTCGATTCCCCCTCTCCCAATATTTACGTTAACATTCCACCAACTTTAGCTCAAGTAAGTATTACAATAATTTGAGGAAACAATGTTTAGGTGTTTTAGTTTAGTGGTTTGGTACTTCAATTGTCACGCGAATCTTTGTTTTCATTTTCGTAAACCAGACACAACAAATTACAAACTAACACTTCAAAAGTAAGGCAGACTGTTGGGAACATGCAGACGAAAAATCAAAAGCAGGATTCCAGGTGGGTAATGTGTTTTGACTATTAGACAATTTTATGCCAGTTGAAAACTGACTTTTCTGCGCATGTGGAAATTGCACATATATATATGAGTGGACATCATCATCTGCAGACAAATCCAGATCCTGTTTCATCATTAGCTTAGCTAAAGTGGAATAGTATGAAGATTACAGCCTAGTAGTTGGTGGAGGCACGAAAGATTACAGCTACGCATGGGAAGTCTCGGTTAATGGGATGCCGGGTCCCCTTTGAGTGTAGAAAAGCTGCTGCTCGACAAATAGGATACCAGCGGAGTCTAACATCCTACGAATAAACCGTTAACGCAGCAGCGCATATATATATGAGTTAGGTTGCCTATGAGTTATTTACACTAAGGTTTTCTACTTTTTCACAAAATTCTTTAAGGTTTTAGAAATTACACAAACACCCCCTGAGGTTTTAAATTGTTTTCACAAAATTCATTTTCATTGATTTTTCAACCAAAGATTGATGGATTTTATACAAAAAAAATTCTCCAAATGACAAAGTTGACCTTTGAGATTGGATTGTAGATACTTTATTGAGGTTAATTTTCTCATTTGCATAAGTGTTTTTTTCAATGAAATCATCAATTTTTGGACAAACAATCAACGAAAAAGGGATTTGTGAAAATAAACTTAAATCTCAGGGGGTGTTCGTGTAATGTTTGAGACATGATGGAAGTTTTGTGAAAACACAAGAAACCTCAGAGGGTGTTAGTGTAAATAGAAATATATTTAATAGTTTGACTGGTAGCTAATTTATGACAGAATTAATAACTGTTGCAATCTTTTAAACTTCGTCACTTTTTGCTTATGTGGATATGAGGCATGCACGTCACTGGCCTGGTAAGGTTTAATTTGATGGTCTCCATGCGGTCGGAGACCCTTTATTTATAATGCTAGGTGGCTTCTGGACGCTTAACTAACAGGCACAAAATAAGCTGGCTGCAAGCATACAACGCTGCCCAAAAGAAAACGGCGCG

Promoter Elements

FIGS. 3A-3E show the various sequence elements identified in thecandidate fruit-specific promoters. Citrus is non-climacteric (i.e., itripens without ethylene and respiration bursts), therefore no ethyleneresponsive element (ERE) sites were identified in any of the citruspromoter sequences, even though ERE is the most common fruit-specificelement.

FIG. 3A presents the sequence elements identified in the citrus promoterCitSEPp. CitSEPp was shown to have a cis-acting element that confershigh transcription levels (5′UTR Py-rich stretch). Other interestingelements identified were the CCGTCC-box and CE3, which are responsiblefor ABA and VP1 responsiveness. Sequence analysis predicted TATA box andCAAT boxes. A summary of the promoter elements identified in CitSEPp isshown in Table 3.

TABLE 3 Promoter elements identified in CitSEPp Element Function 5UTRPy-rich cis-acting element conferring high transcription levels stretchA-box cis-acting regulatory element ABRE cis-acting element involved inthe abscisic acid responsiveness ARE cis-acting regulatory elementessential for the anaerobic induction Box 4 part of a conserved DNAmodule involved in light responsiveness Box I light responsive elementCCGTCC-box cis-acting element involved in ABA and VP1 responsiveness CE3cis-acting element involved in ABA and VP1 responsiveness G Boxcis-acting regulatory element involved in light responsivenessGARE-motif gibberellin-responsive element GCN4_motif cis-regulatoryelement involved in endosperm expression HSE cis-acting element involvedin heat stress responsiveness I-box part of a light responsive elementSkn-1_motif cis-acting regulatory element required for endospermexpression TC-rich repeats cis-acting element involved in defense andstress responsiveness TCA-element cis-acting element involved insalicylic acid responsiveness

FIG. 3B presents the sequence elements identified in the citrus promoterCitWAXp. CitWAXp showed common potential regulatory elements associatedwith hormone, light and stress related responses. The presence of theseputative cis elements indicates that the gene could be regulated byphysiological (hormones) and environmental (light and stress) factors. Asummary of the promoter elements identified in CitWAXp is shown in Table4.

TABLE 4 Promoter elements identified in CitWAXp Element Function ABREcis-acting element involved in the abscisic acid responsiveness ACEcis-acting element involved in light responsiveness ATCT-motif part of aconserved DNA module involved in light responsiveness G-Box cis-actingregulatory element involved in light responsiveness HSE cis-actingelement involved in heat stress responsiveness MBS MYB binding siteinvolved in drought-inducibility O2-site cis-acting regulatory elementinvolved in zein metabolism regulation TCA-element cis-acting elementinvolved in salicylic acid responsiveness TGA element auxin-responsiveelement Box 4 part of a conserved DNA module involved in lightresponsiveness Box I light responsive element Box-W1 fungal elicitorresponsive element AT1-motif part of a light responsive module TC-richrepeats cis-acting element involved in defense and stress responsiveness

FIG. 3C presents the sequence elements identified in the citrus promoterCitUNKp. CitUNKp showed putative hormone responsive elements in thepromoter region, including an ARE motif (involved in the abscisic acidresponsiveness) and a TCA-element (involved in salicylic acidresponsiveness). Also identified were cis-acting elements involved inlight responses including an ATCT motif and a G-box. In addition, thepromoter sequence was found to contain a number of cis-elements relatedto stress responses, including an HSE motif (involved in heat stressresponses), an LTR motif (involved in low-temperature responses), an MBSsite (MYB binding site involved in drought-induction) and TC-richrepeats (involved in defense and stress responses). A summary of thepromoter elements identified in CitUNKp is shown in Table 5.

TABLE 5 Promoter elements identified in CitUNKp Element Function AE partof a module for light response ARE cis-acting regulatory elementessential for the anaerobic induction ATCT-motif part of a conserved DNAmodule involved in light responsiveness G-Box cis-acting regulatoryelement involved in light responsiveness HSE cis-acting element involvedin heat stress responsiveness MBS MYB binding site involved indrought-inducibility O2-site cis-acting regulatory element involved inzein metabolism regulation TCA-element cis-acting element involved insalicylic acid responsiveness TC rich repeats cis-acting elementinvolved in defense and stress responsiveness TGA elementauxin-responsive element Box 4 part of a conserved DNA module involvedin light responsiveness Box I light responsive element Box-W1 fungalelicitor responsive element LTR cis-acting element involved in low-temperature responsiveness P-box gibberellin-responsive element TC-richrepeats cis-acting element involved in defense and stress responsiveness

FIG. 3D presents the sequence elements identified in the citrus promoterCitJuSacp. CitJuSacp showed common potential regulatory elementsassociated with hormone, light and stress related responses. A summaryof the promoter elements identified in CitJuSacp is shown in Table 6.

TABLE 6  Promoter elements identified in CitJuSacp Element FunctionStrand Position Sequence ABRE cis-acting element +   61 CACGTGinvolved in the abscisic acid  responsiveness ARE cis-acting   − 1098TGGTTT regulatory element essential for  the anaerobic  induction ATCT-part of a  −  944 AATCTAATCT motif conserved DNA  (SEQ ID module involved NO: 32) in light responsiveness G-Box cis-acting   + -61, CACGTG, regulatory element 1071 CACGTT involved in lightresponsiveness HSE cis-acting element +  245 AAAAAATTTCinvolved in heat  (SEQ ID  stress NO: 33) responsiveness MBSMYB binding site − − 261, CAACTG, involved in  933 TAACTG drought-inducibility O2-site cis-acting   − − 1080 GATGACATGG regulatory element(SEQ ID  involved in zein  NO: 34) metabolism  regulation TCA-cis-acting element + 1201 GAGAAGAATA element involved in  (SEQ ID salicylic acid  NO: 35) responsiveness TGA auxin-responsive −  907AACGAC element element

FIG. 3E presents the sequence elements identified in the plum promoterPfeMybAp.

PfeMybAp was shown to contain putative cis elements such as MBSI, MREand an as-2-box involved in flavonoid and light responsive signaling. Asummary of promoter elements identified in PfeMybAp is shown in Table 7.

TABLE 7 Promoter elements identified in PfeMybAp Element FunctionATCT-motif part of a conserved DNA module involved in lightresponsiveness ARE cis-acting regulatory element essential for theanaerobic induction ABRE cis-acting element involved in the abscisicacid responsiveness G-Box cis-acting regulatory element involved inlight responsiveness HSE cis-acting element involved in heat stressresponsiveness MBS MYB binding site involved in drought-inducibilityBox-W1 fungal elicitor responsive element Box 4 part of a conserved DNAmodule involved in light responsiveness CAG-motif part of a lightresponse element MBSI MYB binding site involved in flavonoidbiosynthetic genes regulation MRE MYB binding site involved in lightresponsiveness as-2-box involved in shoot-specific expression and lightresponsiveness

Conclusion

Taken together, the results provide successful isolation of theidentified fruit-specific promoters and characterization of theirsequences. Identification of the various regulatory elements of thecandidate promoters from this study promotes a better understanding ofmechanisms underlying gene expression specificity in fruit.

Example 3: Functional Characterization of the Fruit-Specific PromotersUsing Agrobacterium-Mediated Transient Expression Assay

The following example describes the functional analysis of thefruit-specific promoters using an Agrobacterium-based transientexpression assay.

Materials and Methods

Molecular Constructs

The Promoter::GUSPlus molecular constructs used in this study are asdescribed in Example 2. The vector contains the GUSPlus and Nosterminator sequences, as well as the attP and res recombination sitesfor future recombinase mediated construct exchange (RMCE) genomictargeting. The attP and res are recombinase recognition sites for theBxb1 and CinH recombinase enzymes respectively. LB and RB designate theAgrobacterium left border and right borders, respectively. The vectoralso contains the nptII gene for kanamycin selection. In addition to thecitrus and plum candidate promoter sequences, two fruit-specificpromoters from tomato, E8p and PGp, along with the constitutive promoter35Sp were used as controls for this study.

Agrobacterium Suspension

Agrobacterium cultures (5 mL) were grown overnight from individualcolonies at 28° C. in LB medium plus selective antibiotics, transferredto 50 mL induction medium (0.5% beef extract, 0.1% yeast extract, 0.5%Peptone, 0.5% Sucrose, 2 mM MgSO4, 20 mM acetosyringone, 10 mM MES, pH5.6) plus antibiotics, and grown again overnight. Next day, cultureswere recovered by centrifugation, resuspended in infiltration medium (10mM MgCl2, 10 mM MES, 200 mM acetosyringone, pH 5.6; optical density 0.5to 1.0 unless stated otherwise), and incubated at room temperature withgentle agitation (20 rpm) for a minimum of 2 h. Cultures were combinedwhen required, collected with a syringe, and injected into fruits asdescribed below.

Agrobacterium Injection (Agroinjection)

Tomato fruits (Solanum lycopersicum cv Micro-Tom) were infiltrated usinga 1-mL syringe with a 0.5-316-mm needle (BD Pastipak). Needle wasintroduced 3 to 4 mm in depth into the fruit tissue through the stylarapex, and the infiltration solution was gently injected into the fruit.The total volume of solution injected varied with the size of the fruit,with a maximum of 600 mL in mature green tomatoes. The progress of theprocess could be followed by a slight change in color in the infiltratedareas. Once the entire fruit surface has been infiltrated, some drops ofinfiltration solution begin to show running off the hydathodes at thetip of the sepals. Only completely infiltrated fruits were used in theexperiments.

GUS Staining

β-glucuronidase (GUS) was detected using a GUS staining solution (0.1 Msodium phosphate pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassiumferricyanide, 1.5 g/L X-Gluc, and 0.5% v/v Triton X-100) generally for 4to 20 h at 37° C. The incubation time was adjusted based on the strengthof the staining observed. After staining, green tissues were passedthrough several changes of 70% and 95% ethanol to remove chlorophyll.

Imaging

The Agroinjection images in petri plates were observed and photographedin a Leica MZ16-F (Leica Microsystems, Inc., Buffalo Grove, Ill.) stereozoom light microscope equipped with a Qlmaging Retiga 2000 R fastcooled, digital color camera.

Results

To investigate the ability of the candidate promoter fragments to confergene activity, the Agrobacterium-mediated transient expression assay wasperformed in tomato fruit tissue. Tomato fruits at mature green and ripestages (22-25 d after anthesis) were Agroinjected using Agrobacteriumsuspensions containing the previously described Promoter::GUSPlusmolecular constructs (FIG. 3). Agroinjected fruits were then treatedwith GUS staining solution and visualized for qualitative analysis ofthe GUS expression, which reports the activity of the candidatepromoter.

Candidate Promoters Drive Expression of the GUS Reporter Gene in Fruit

GUS staining patterns of the Agroinjected tomato fruits are shown inFIG. 4. From the results, high levels of GUS activity (blue staining)were detected in fruits 4 d after Agroinjection. GUS expression was notdetected in wild type tomato fruit Agroinjected with empty vectorcontrol, whereas strong and varied expression patterns were detected infruits injected with various candidate promoter constructs. Differentcandidate promoters showed different intensities of GUS expression inimmature versus ripe fruits. Similar amounts of Agrobacterium wereinjected into each fruit tested. CitSEPp lines showed very weakexpression both in immature and ripe fruits; the staining was mostly themucosal sack around the seeds. CitWAXp lines showed strong expressionboth in immature and ripe fruits. CitUNKp gave rise to the strongest GUSexpression among all fruits both in young immature tomato fruits as wellas in mature ripe fruits. CitJuSacp lines showed stronger expression inmature fruits compared to immature fruits, whereas PfeMybAp lines showedsimilarly strong expression in both unripe and ripe fruits.

Conclusion

Together, these observations demonstrated that the candidate promoterssuccessfully drove expression of reporter gene in tomato fruit tissuevia Agroinjection, indicating that the promoter candidates from citrusand plum contain active promoter elements regulating GUS expressionpatterns in tomato fruit.

Example 4: Functional Characterization of the Fruit-Specific PromotersUsing Stable Transgenic Tomato Plants

The following example illustrates the functional analysis of thefruit-specific promoters using stable transgenic tomato plants.

Materials and Methods

Transgenic Tomato Plants

Transgenic Micro-Tom Rg1 tomato plants were produced usingpCTAGII-derived binary vectors (GenBank accession number MG818373) andkanamycin selection with an established Agrobacterium-mediatedtransformation method (Pino et al., 2010).

Droplet Digital PCR

Genomic DNA was extracted by grinding a 1-cm² piece of tomato leaf in400 μL of buffer (200 mm Tris-HCl pH 7.8, 250 mm NaCl, 25 mm EDTA, 0.5%SDS). After centrifugation and isopropanol precipitation, the pellet waswashed with 70% ethanol and resuspended in 50 μL of water with 1 mMRNase A. PCR amplification was performed using 2 μL of genomic DNA inreactions with a total volume of 25 μL. Presence of the transgene wasconfirmed by PCR using transgene specific primers. Droplet digital PCR(ddPCR) was performed following the methods in Collier et al., The PlantJournal (2017) 90, 1014-1025. Sequences of the ddPCR primers and probesthat were used for detecting reference gene (CsUBC) are:

CsUBC_ddPCR-F2: (SEQ ID NO: 18) 5′-CGCTCAGGTGATATAAGAGG-3′,CsUBC_ddPCR-R2: (SEQ ID NO: 19) 5′-TGAATAGGGCTTCGTCAATC-3′,  andCsUBC probe:  (SEQ ID NO: 20) 5′-AAGGATGTACACTAGACTTGCGGC-3′.

GUS Staining

β-glucuronidase (GUS) was detected as using a GUS staining solution (0.1M sodium phosphate pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mMpotassium ferricyanide, 1.5 g/L X-Gluc, and 0.5% v/v Triton X-100)generally for 4 to 20 h at 37° C. The incubation time was adjusted basedon the strength of the staining observed. After staining, green tissueswere passed through several changes of 70% and 95% ethanol to removechlorophyll.

Imaging of Transgenic Plants

The photographs of the plants were recorded using a Nikon D7000 digitalcamera with an AF Micro Nikkor 60 mm 1:2.8 D lens or AF-S Nikkor 18-70mm DX lens (Nikon Inc., Melville, N.Y.) under tungsten lamps (Philips,120 V, 300 W). The camera was set manually for all parameters includingISO sensitivity, focus, f-stop and time. A photography gray card wasused as a reference to get the correct exposure.

Quantitative GUS Expression Assay

GUS expression was quantitated using the method described below and inCold Spring Harb. Protoc. (Blazquez, 2007), Arabidopsis: A Lab. Manual.Cold Spring Harb. Lab. Press, (Weigel and Glazebrook, 2002), andJefferson et al., EMBO J (1987) 6:3901-3901. The expression ofβ-glucuronidase (GUS) can be accurately determined in extracts of planttissue using 4-methylumbelliferyl β-D-glucuronide (4-MUG) as asubstrate. Upon hydrolysis by GUS, the fluorochrome 4-methylumbelliferone (4-MU) is produced. Using excitation at 365 nm andmeasuring emission at 455 nm, the amount of 4-MU produced can bequantified. Under these conditions, background fluorescence from thesubstrate is negligible, especially if the appropriate filter isselected.

Results

Generation of Transgenic Tomato Lines

A total of 15-20 independent T0 transgenic lines were obtained for eachpromoter construct using the Agrobacterium-mediated transformationmethod and were grown to maturity in a greenhouse. All tissue culturewas done on kanamycin selection. The individual lines were PCR testedand were found to be kanamycin positive.

The individual lines were grown to maturity, with overall growth anddevelopment monitored. Compared to wild type (WT), the transgenic plantsshowed no significant difference in either vegetative or reproductivegrowth patterns, with the exception of the CitJuSacp lines (FIG. 5).Based on the kanamycin positivity and initial GUS analysis on vegetativeand reproductive tissues, seeds were collected from selected T0 linesfor further analysis in the T1 generation.

Seedless Fruit Phenotype of the CitJuSacp Transgenic Lines

Seed and fruit development are intimately related processes controlledby internal signals and environmental cues. Interestingly, the CitJuSacptransgenic lines generated seedless fruits even though the fruitdevelopment was similar to that of WT. The CitJuSacp lines' fruits hadmore biomass and were larger in size compared to WT. Compared to WT, thetransgenic tomato lines either did not develop seeds or developed smallnon-viable seeds (FIG. 6 and FIG. 7). However, upon repeating transgenicproduction using this same construct in Arabidopsis and tobacco, theseedless phenotype was not detected. The seedless nature of theCitJuSacp tomato lines may be a very specific interaction between theCitJuSacp and the tomato genome that will require further study.

A genetic method for obtaining seedless fruits is valuable for themarket only if fruit quality and productivity are not curtailed. In thiscase, both the quality and productivity were not compromised.Furthermore, these plants did not display any vegetative or reproductivealterations except for the lack of seeds. Therefore, the seedless fruitphenotype observed in the CitJuSacp transgenic lines has valuableimplications in developing seedless fruit cultivars. The nucleotidesequence of the CitJuSacp is included herein as SEQ ID NO: 4 or acertain degree of sequence identity to SEQ ID NO: 4.

Trans Gene Copy Number Characterization

T1 plants were germinated on kanamycin-containing media and thentransferred to soil. Droplet digital PCR (ddPCR) was conducted toidentify low-copy lines in T1 generation (Table 8 and Table 9). TheddPCR assay can determine transgene copy number in independent events,as well as can be used to distinguish hemizygous lines from theirhomozygous siblings. The tomato single-copy gene prosystemin (McGurl etal., 1992) was previously utilized in qPCR experiments as an endogenousreference for transgene copy number measurement (Collier et al., 2017).Five independent transgenic events were examined. Hemizygous andhomozygous individuals were confidently identified that carrysingle-copy nptII transgene insertions. For each candidate promoterconstruct, three representative lines were selected based on ddPCRtransgene copy number results and initial GUS analysis in T0 generationfor further study (Table 8). ddPCR results indicate that the CitJuSacplines only had a half or single copy of the transgene in all transgenicplants produced. These results were confirmed by the COT analysis fortotal genomic content when compared against WT (FIG. 7), which suggestedthat the CitJuSacp lines had undergone complete genome duplication.

TABLE 8 Transgene copy number of the candidate promoter transgenictomato plants CitWAXp CitUNKp CitSEPp PfeMybAp CitJuSacp Wild typeTransgenic Plants Transgenic Plants Transgenic Plants Transgenic PlantsTransgenic Plants control Line Copy Line Copy Line Copy Line Copy LineCopy Line Copy WAX1-1 3.00 UNK2-1 0.98 SEP4-1 1.94 Pfe1-1 0.98 JS2-10.51 WT1-1 0.02 WAX1-2 5.91 UNK2-2 1.98 SEP4-2 0.97 Pfe1-3 1.01 JS2-40.98 WT1-2 0.03 WAX1-3 2.96 UNK2-3 1.01 SEP4-3 1.01 Pfe1-4 2.01 JS2-60.51 WAX1-4 5.00 UNK2-4 1.94 SEP4-5 1.00 Pfe1-5 0.97 JS2-7 0.97 WAX1-72.09 UNK2-5 1.01 SEP4-6 1.00 Pfe1-6 1.95 JS2-8 0.94 WAX3-1 2.92 UNK3-12.04 SEP8-1 4.98 Pfe2-2 1.00 JS2-9 1.01 WAX3-3 6.10 UNK3-2 1.03 SEP8-25.05 Pfe2-3 1.94 JS4-1 1.01 WAX3-5 5.00 UNK3-3 0.97 SEP8-4 1.92 Pfe2-40.99 JS4-2 0.50 WAX3-6 2.90 UNK3-4 1.01 SEP8-5 2.00 Pfe2-5 1.96 JS5-10.52 WAX3-7 2.94 UNK3-5 2.02 SEP8-6 5.09 Pfe2-6 2.02 JS5-2 0.53 WAX5-16.04 UNK4-1 1.00 SEP10-1 1.01 Pfe9-1 1.90 JS5-3 0.49 WAX5-2 2.27 UNK4-21.00 SEP10-2 1.03 Pfe9-3 0.98 JS5-4 0.51 WAX5-3 3.05 UNK4-3 1.99 SEP10-31.90 Pfe9-4 1.92 JS5-6 0.02 WAX5-4 1.04 UNK4-4 2.01 SEP10-4 1.91 Pfe9-51.00 JS7-1 1.90 WAX5-9 2.82 UNK4-5 1.00 SEP10-6 1.04 Pfe9-6 0.99

TABLE 9 Transgene copy number of the control promoter transgenic tomatoplants PGp Transgenic E8p Transgenic 35Sp Transgenic Plants PlantsPlants Line Copy Line Copy Line Copy PG1-1 0.97 E81-1 13.80 35S1-3 0.95PG1-2 1.89 E81-2 10.98 35S1-4 0.91 PG1-3 0.95 E81-5 13.90 35S1-6 0.92PG1-4 0.94 E81-6 18.10 35S1-7 1.04 PG1-5 1.92 E81-9 10.80 35S1-8 1.97PG3-3 2.81 E82-3 2.84 35S2-2 0.90 PG3-4 0.91 E82-4 5.07 35S2-3 1.97PG3-5 2.82 E82-7 3.02 35S2-4 0.90 PG3-6 1.91 E82-8 5.90 35S2-6 0.92PG3-8 2.16 E84-1 2.04 35S2-8 0.97 PG6-4 1.03 E84-2 2.04 35S3-1 0.92PG6-5 1.07 E84-4 1.99 35S3-4 1.88 PG6-6 1.06 E84-3 1.00 35S3-5 1.85PG6-7 1.09 E86-1 13.30 35S3-6 1.89 PG6-8 1.09 E86-2 14.20 35S3-7 2.07PG7-1 1.00 E86-4 11.20 PG7-2 1.10 E86-5 11.80 PG7-3 1.90 E86-6 15.10PG7-4 1.00 PG7-5 1.04 PG8-2 12.20 PG8-3 11.80 PG8-4 12.00 PG8-6 13.10PG8-7 11.09

Qualitative Analysis of Promoter Activity in Transgenic Tomato

Qualitative analysis of promoter activity was conducted by comparingintensity of GUS staining between promoter transgenic lines andcontrols. Images showing GUS staining in various vegetative andreproductive tissues are presented in FIGS. 8A-8B. A summary of thetissue-specific expression patterns for the candidate promoters areshown in Table 10.

TABLE 10 Summary of qualitative GUS analysis in transgenic tomatoImmature Mature Promoter::GUS Seedling Leaf Stem Root Flower fruit fruit35Sp::GUS Yes Yes Yes Yes Yes Yes Yes E8p::GUS Yes (faintly Yes (faintlyNo Yes Yes Yes Yes leaky leaky (leaky) (leaky) sometimes) sometimes)PGp::GUS Yes (faintly No No No No Yes, Yes, leaky weak strong sometimes)CitSEPp::GUS No No No No No No Yes (most strong in seeds) CitWAXp::GUSYes (weak, Yes (faintly No No Yes Yes Yes mostly in leaky leaves)sometimes) CitUNKp::GUS Yes Yes Yes Yes Yes Yes Yes (leaky) (leaky)(leaky) CitJuSacp::GUS Yes (leaky Yes (faintly No No Yes Yes Yessometimes) leaky (leaky) sometimes) PfeMybAp::GUS Yes (leaky Yes (leakyNo No Yes Yes Yes sometimes) in midrib)

As expected, WT plants did not display any GUS staining in eithervegetative or reproductive tissues, while the positive controltransgenic lines transformed with 35Sp promoter fused to reporter geneshowed GUS staining in all tissues. The known fruit-specific tomato E8ppromoter showed strong activity in both unripe and ripe fruits, leakyactivity in flower, and leaky activity in seedling and mature leaf.Another known fruit-specific PGp promoter from tomato showed minor leakyactivity in seedling but no activity in mature leaf, root or flower.However, activity of PGp promoter was very strong in ripe fruit comparedto that in young immature fruit, presumably due to the involvement ofthe PG gene in fruit ripening process.

CitSEPp transgenic lines showed no GUS expression in leaf and petiole,no expression in root and flower, no expression in mature leaf, veryfaint expression in young immature fruit, but strong expression inmature fruit. The expression in the fruit was strongest in the mucosalsac surrounding the seeds, followed by locular tissue, pericarp tissueand placental tissue.

CitWAXp transgenic lines showed some weak leaky GUS expression in leafand flower, no expression in the root and mature leaf, but strongexpression in both immature and immature fruits.

CitUNKp transgenic lines showed weak GUS expression in seedling andmature leaf, and leaky expression in stem, root, and flower. Immaturefruit showed stronger expression than mature fruit. The expression inthe fruit was strongest in the outer epidermis and seeds, followed bylocular tissue, pericarp tissue and placental tissue.

CitJuSacp transgenic lines showed some weak faint GUS expression inseedling, no expression in mature leaf, no expression in root and flowerbut strong expression in young immature fruit and ripe fruit. Theexpression in the fruit was strongest in the locular tissue, pericarptissue, and placental tissues in the ripe fruit. Seed development wasseverely affected in the transgenic lines (seedless).

PfeMybAp transgenic lines showed an interesting pattern of GUSexpression in leave that had blue stains in the mid-rib and the base ofthe leaf, while the root did not show any blue staining. The flower wasmostly stained in the petals. Both unripe and ripe fruits, however, hadvery strong GUS expression throughout the fruit.

Quantitative Analysis of Promoter Activity

To confirm the results of the qualitative analysis, quantitativeanalysis of promoter activity was conducted using the quantitative GUSexpression assay (Jefferson et al., 1987, Weigel and Glazebrook, 2002,and Blázquez, 2007). Quantitative GUS expression assay was performed inleaf, immature fruit, and ripe fruit of three representative transgenictomato lines for each Promoter::GUS construct.

Results of the quantitative GUS expression assay further confirmed thepromoter activity patterns concluded from the qualitative analysis. Asummary of the quantitative GUS expression results is shown in FIGS.9A-9D. WT plants did not display GUS staining in any tissue. Linestransformed with construct of positive control 35S promoter fused toreporter gene showed high level of GUS expression in all tissues asexpected. CitSEPp transgenic lines showed GUS expression only in ripefruits. CitWAXp transgenic lines showed some expression in leaves andhigher expression in ripe fruits. CitUNKp transgenic lines showed highlevels of expression in leaves compared to WT. Expression was alsodetected in both unripe and ripe fruits. CitJuSacp transgenic linesshowed more fruit preferential expression compared to WT. PfeMybAptransgenic lines showed fruit preferential expression compared toleaves.

From the results, the citrus candidate promoter CitSEPp had the bestfruit-specific expression pattern in tomato. Among all the candidatecitrus promoters tested, CitSEPp was the only one showing GUS expressionin the seeds and surrounding tomato pulp in ripe fruits only (FIGS.8A-8B and FIGS. 9A-9D). Specifically, CitSEPp in tomato showed no GUSexpression in leaves and petiole, no expression in roots and flowers, noexpression in mature leaves, very faint expression in young immaturefruits but strong expression in mature fruits. The expression in thefruit was strongest in the seeds followed by locular tissue, pericarpand placental tissues. The qualitative analysis correlates with thequantitative data as shown in the graph. Compared to WT, GUS activitywas detected only in the ripe fruits of the CitSEPp::GUS lines (FIG.9D).

Based on the above analysis, transgenic Arabidopsis and tobaccotransgenic lines were generated. FIG. 10 shows GUS staining of T1transgenic lines on Arabidopsis seedling (top two images, no staining)and tobacco leaf and flower (middle two images, no staining), ascompared to tomato (bottom images, staining in fruit only, as previouslypresented in FIG. 8A). The results confirmed that CitSEPp has a highfruit-specific expression pattern in Arabidopsis and tobacco as intomato.

Conclusion

Taken together, the results indicate that the candidate promoters arefunctional in driving gene expression in plants with strong fruitpreference, where the citrus candidate promoter CitSEPp has thestrongest fruit-specific expression pattern in tomato. Additionally, itwas found that CitJuSacp transgenic lines generated seedless fruits withotherwise normal development, which provides significant implications inthe development of seedless fruit varieties.

Example 5: Modification of Anthocyanin Accumulation Using Fruit-SpecificPromoters

The following example illustrates the use of the candidate promoters inmodifying anthocyanin accumulation in fruit. Plants produce a group ofmetabolites, such as flavonoids, that belong to a group of plant naturalproducts, playing an important role in protection against variousstresses. Anthocyanin forms the largest sub-class of flavonoidsconferring different colors in fruits and flowers. Increased anthocyanincontent is a desired commercial fruit trait. The objective of this studywas to use the identified fruit-specific promoters for manipulation ofthe anthocyanin metabolic pathway to enable the generation of new plantvarieties with improved fruit quality.

Methods and Materials

Molecular Constructs

The fruit-specific promoters, together with the control promoter (i.e.tomato E8 promoter E8p), were PCR amplified, digested, and cloned intopCTAG6-GUSPlus vector (deposited in GenBank at NCBI, accession numberMG836292). For transgenic Arabidopsis and tobacco plants, thePromoter::MoroMybA constructs were inserted into the pCTAG6-GUSPlusvector backbone for transformation. For transgenic citrus plants, inaddition to the Promoter::MoroMybA constructs, a Nosp::AtFT::NosTconstruct was inserted into the pCTAG6-GUSPlus vector backbone at thePspOMI site for transformation in order to shorten the flowering time ofthe transgenic citrus plants. Nosp has a nucleotide sequence of SEQ IDNO: 21. AtFT has a nucleotide sequence of SEQ ID NO: 22. NosT has anucleotide sequence of SEQ ID NO: 23. A diagram showing the molecularconstructs is presented in FIG. 11.

Transgenic Arabidopsis Plants

Agrobacterium tumefaciens strain GV3101 was used for transformation ofArabidopsis ecotype Ler by the floral dip method (Clough and Bent 1998)that is modified by adding 0.01% Silwet L-77 (Lehle Seeds, Round Rock,Tex.) to the infiltration medium. Primary transformants were selected onMS medium (Sigma, St. Louis, Mo.), 1% sucrose, 0.7% agar with 20 μg/mlhygromycin or 50 μg/ml kanamycin as needed for 10 days prior tocultivation in soil.

Trans Genic Tobacco Plants

Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) was used for leafdisc transformation (Horsch and Klee 1986). Agrobacterium cells weregrown to an optical density of 1.0 at 600 nm (OD600), and a finalsuspension at OD600 of 0.5 was used for plant infection. Young, healthygreen leaves were cut into pieces approximately 10 mm in length, and theleaf segments were incubated in an Agrobacterium suspension for 30minutes. The leaf segments were then blotted dry on sterile filter paperfor 5 min and placed onto MS co-cultivation medium (Sigma, St. Louis,Mo.) in sterile Petri dishes and kept in the growth chamber at 25° C.for three days in the dark. The infected leaf explants were thentransferred to regeneration/selection medium (24° C. with 16 h of lightand 8 h of dark at 20° C.). Primary tobacco transformants were selectedon MS medium, with 100 mg/mL kanamycin. After 2-3 weeks, the infectedleaf explants were transferred onto fresh regeneration/selection media.Separate shoots from explants were excised carefully and transferredinto plant culture dishes containing rooting medium. Rooted plants weregrown in Sunshine potting mix (Sun Gro Horticulture Ltd. Agawam, Mass.)in the greenhouse with 16 h of light at 150 photosynthetic photon fluxdensity (μmol photons m-2 s-1) at 23° C. and 8 h of dark at 20° C. with70% humidity. Twenty kanamycin resistant lines were obtained from the T₀generation for each construct. T₁ seeds were collected and selected onan MS plate supplemented with kanamycin 100 mg/mL.

Trans Genic Citrus Plants

The transgenic Carrizo citrange and Mexican lime citrus lines weregenerated using epicotyl explants protocol as previously described by deOliveira et al., Plant Cell Rep (2009) 28:387-395, and de Oliveira etal., HortTechnology (2016) 26(3), 278-286.

Semi-Quantitative PCR (Reverse Transcription-PCR, RT-PCR)

Leaf tissues were collected from plants grown in greenhouse andhomogenized using the MagNA Lyser instrument (Roche Life Science). RNAwas extracted using the sample preparation kit Direct-zol RNA MiniprepPlus (Zymo Research, catalog number R2071). cDNA synthesis was performedusing the Revert Aid H minus First Strand cDNA Synthesis Kit for 100rkns(Thermo Fisher Scientific, catalog number K1632). PCR assay for gene ofinterest (e.g. AtFT) was performed using CitEF1a as the internalcontrol. PCR products were loaded into 1.2% agarose gel for gelelectrophoresis. Gel image was analyzed using the image processingsoftware ImageJ (Schneider et al., Nature methods (2012) 9(7): 671:675).The resulting data of gel band intensity was used for calculating therelative expression of the gene of interest compared to the internalcontrol.

Droplet Digital PCR

Genomic DNA was extracted by grinding a 1-cm² piece of tobacco leaf in400 μL of buffer (200 mm Tris-HCl pH 7.8, 250 mm NaCl, 25 mm EDTA, 0.5%SDS). After centrifugation and isopropanol precipitation, the pellet waswashed with 70% ethanol and resuspended in 50 μL of water with 1 mMRNase A. PCR amplification was performed using 2 μL of genomic DNA inreactions with a total volume of 25 μL. Presence of the transgene wasconfirmed by PCR using transgene specific primers, including NptII_F2819with a nucleotide sequence of 5′-TTGCCGAATATCATGGTGGA-3′ (SEQ ID NO:24), NptII_R2931 with a nucleotide sequence of5′-TCAGCAATATCACGGGTAGC-3′ (SEQ ID NO: 25), SISys_Probe with anucleotide sequence of 5′-TGCAACATCCTTCTTTCTTCTCGTG-3′ (SEQ ID NO: 26),SISys_F with a nucleotide sequence of 5′-GCAATATCAAGAGCCCCGTC-3′ (SEQ IDNO: 27), SISys_R with a nucleotide sequence of 5′-ATGTGTGCTAAGCGCTCC-3′(SEQ ID NO: 28), codA_ddPCR_F1 with a nucleotide sequence of5′-CGGGCAGATTAACGATGG-3′ (SEQ ID NO: 29), codA_ddPCR_R1 with anucleotide sequence of 5′-CGCATCAAACCCATTTTCAG-3′ (SEQ ID NO: 30), andcodA Probe with a nucleotide sequence of 5′-CGGCAGGATAATCAGGTTGGC-3′(SEQ ID NO: 31). Droplet digital PCR (ddPCR) was performed following themethods in Collier et al., 2017.

Results

The MybA gene is the key player activating the anthocyanin metabolicpathway. The MoroMybA is a synthetic version of the citrus CsRuby(Butelli et al., 2012) coding sequence. MoroMybA encodes a 262-aminoacid protein and was used for transgenic citrus production and MybAtransgene studies as described in Dasgupta et al., 2017. In addition,the 35Sp-MoroMybA construct was shown to be functional in tobacco (FIG.12).

Based on the promoter expression analyses in the preceding examples,three citrus fruit-specific promoters (CitWAXp, CitUNKp, CitJuSacp) werechosen and fused to MoroMybA gene to make an all-citrus transgeneconstruct. In addition, the tomato control promoter E8p and candidateplum promoter PfeMybAp were fused to MoroMybA for stable transgenictesting. These Promoter::MoroMybA(MM) constructs form a novel moleculartoolbox that facilitates the expression of traits specifically withinfruit tissue of desired transgenic plants.

For citrus transgenic plants, there is an additional consideration. Mostcitrus trees need 5-15 years to begin flowering and fruiting. The longjuvenile phase delays regular fruit production for years. Alternatively,early flowering has been achieved in transgenic trees, including citrus,by constitutively over-expressing flower meristem identity genes.FLOWERING LOCUS T gene (FT) is a key regulator of the floweringtransition. It has already been shown that the FT overexpression systeminduces an extremely early fruiting phenotype and two fruiting cyclesper year in sweet orange plants (Pons et al., 2013). Considering theabove facts, for citrus transgenic plant generation, the Arabidopsis FTgene (AtFT) was added into the construct to reduce the flowering timeperiod and quickly assess citrus fruits accumulating anthocyanin.

Phenotypic Analysis of Anthocyanin Accumulation in TransgenicArabidopsis Lines

Selected Promoter::MoroMybA constructs without the AtFT gene were usedfor Arabidopsis floral dip transformation. A total of 12-15 independentT1 kanamycin positive lines were generated for each construct and wereanalyzed for the anthocyanin accumulation phenotype. For each construct,three representative T1 lines were checked for heritability of phenotypein T2 generation.

Results showed that WT Arabidopsis lines did not show any anthocyaninaccumulation whereas 35Sp::MoroMybA showed anthocyanin accumulation asexpected in seeds, seedlings, young leaves, flowers and siliques.CitWAXp::MoroMybA and CitJuSacp::MoroMybA transgenic Arabidopsis linesshowed anthocyanin accumulation in germinating seedlings, flowers,siliques and seeds but not in matured leaves and stems. Anthocyanin wasnot detected in any vegetative tissue of CitUNKp and PfeMybAp transgeniclines. However, these lines accumulated anthocyanin in seeds only.Tomato fruit-specific promoter E8p was used as a positive control forthis study to be fused to MoroMybA, and the resulting transgenic linesalso showed anthocyanin accumulation only in seeds. A summary of theArabidopsis anthocyanin accumulation analysis on a representative linefor each construct is shown in FIG. 13.

Phenotypic Analysis of Anthocyanin Accumulation in Transgenic TobaccoLines

Selected Promoter::MoroMybA constructs without AtFT gene were used fortobacco transformation. A total of 12-15 independent T0 kanamycinpositive lines were generated for each construct and were analyzed forthe anthocyanin accumulation phenotype. For each construct, threerepresentative lines were checked for heritability of phenotype in T1generation.

Expression of the 35Sp::MoroMybA construct in tobacco displayed auniform and homogenously consistent light purplish coloration throughoutthe entire plant compared to WT. The specificity of expression conferredby the construct CitWAXp::MoroMybA was examined in several tissues andorgans of the transgenic tobacco plants. Anthocyanin was not detected inthe any vegetative tissue. Anthocyanin accumulation was detected in theseedpod outer layer as well as the seeds of transgenic tobacco lines,suggesting a highly fruit-specific expression pattern of the CitWAXppromoter.

The specificity of expression conferred by the constructCitUNKp::MoroMybA was examined in several tissues and organs of thetransgenic tobacco plants. Anthocyanin was not detected in anyvegetative tissue. Anthocyanin accumulation was only detected in theseeds of transgenic tobacco lines, suggesting a highly fruit specificexpression pattern of the CitUNKp promoter. There was no expression onthe outer seedpod cover. No accumulation was detected in vegetativetissues or flower.

The specificity of expression conferred by the constructCitJuSacp::MoroMybA was examined in several tissues and organs of thetransgenic tobacco plants. Anthocyanin was not detected in anyvegetative tissue. Anthocyanin accumulation was detected in stigma offlowers, seedpod outer and inner layer, as well as seeds of thetransgenic tobacco lines.

For the PfeMybAp::MoroMybA lines, anthocyanin was detected in the youngseeds in seedpod as well as in mature seeds. No accumulation ofanthocyanin was detected in vegetative tissues or flower.

For the E8p::MoroMybA lines, anthocyanin was detected only in matureseeds. Accumulation of anthocyanin was not detected in vegetativetissues, flower or immature seeds.

A summary of the tobacco anthocyanin accumulation analysis on arepresentative line for each construct is shown in FIG. 14.

Phenotypic Analysis of Flowering Time in Transgenic Citrus Lines

Fruit and forest trees have a long juvenile period, during which noreproductive development occurs. In citrus, the juvenile period rangesfrom 5-15 years, which has hampered traditional breeding and geneticstudies. Therefore, AtFT was added to the vector constructs for citrusplants to reduce the time to flower and more quickly assess citrusfruits accumulating anthocyanin.

Agrobacterium-mediated transformation of Mexican lime explants resultedin the production of a large number of putative kanamycin-resistanttransgenic plants. The constructs used for transformation are shown inFIG. 11 with selected Promoter::MoroMybA::AtFT constructs (promotersCitWAXp, CitUNKp, CitJuSacp, PfeMybAp and E8p). There was no differencein the ability to regenerate shoots following co-cultivation andincubation with any of the designed vectors. All plants grew normallyand there was no phenotypic abnormality observed between transgenicplants and non-transgenic controls. Rooted plants developed slowly inthe first six months of transferring to soil, followed by rapid plantgrowth. 10-15 transgenic lines from each construct were hardened off inthe greenhouse. ddPCR was conducted to identify 1-2 copy lines. Geneticanalysis of citrus species is challenging, while traditional methodslike Southern blot can be difficult or time taking. Droplet digital PCR(ddPCR) serves as an efficient, faster method for identifyingsingle-copy transgene insertion events from a population of transgeniclines. In this study, transgenic citrus lines with a single copy weresuccessfully determined with ddPCR (Table 11).

TABLE 11 Days to flower and copy number comparison Date of Age in Dateof transfer Copy weeks during Line flowering from RM number flowering JS7-2 Nov. 24, 2017 Aug. 16, 2016 2 65 JS 7-15 Jun. 14, 2017 Dec. 19, 20153 77.6 JS 7-19 May 31, 2017 Jan. 20, 2016 4 71 JS 7-23 Nov. 24, 2017Apr. 21, 2016 1 83 WAX 9-3 Jun. 7, 2017 Dec. 29, 2015 4 75.1 WAX 9-9Nov. 17, 2017 Aug. 16, 2016 4 65 WAX 9-14 Nov. 16, 2017 Apr. 21, 2016 382 WAX 9-6 Feb. 26, 2018 Aug. 16, 2016 3 82 UNK 4-3 May 31, 2017 Dec. 2,2015 3 78 PFE 5-1 Jul. 17, 2017 Dec. 2, 2015 3 85 PFE 5-2 Jul. 17, 2017Dec. 2, 2015 3 85 PFE 5-3 Jul. 17, 2017 Dec. 2, 2015 1 85.7 PFE 5-4 Aug.10, 2017 Dec. 2, 2015 1 88 E8 10-4 Aug. 9, 2017 Oct. 29, 2015 5 92 E810-9 Aug. 9, 2017 Dec. 29, 2015 2 84.1 E8 10-11 Jun. 14, 2017 Dec. 29,2015 1 76.1 E8 10-24 Feb. 26, 2018 Apr. 21, 2016 4 96

RT-PCR was done on transgenic Mexican lime lines to test the AtFTexpression as compared to the internal control CitEF1a on selectedlow-copy (preferably single-copy) lines in T0 generation (FIGS.15A-15E). The two negative controls were one with no reversetranscriptase enzyme added during cDNA synthesis and the other with nocDNA added during PCR. A band size of 500 bp was expected for AtFTexpression, using gene-specific primers that amplifies the full cDNA ofAtFT used in the construct (lower panels of the figures). All reactionswere done at the same time. The upper panels of the figures show inhistograms the AtFT expression relative to CitEF1a (Y-axis) on lineswith different copy numbers (CN, X-axis) for each construct tested.Based on the results, transgenic Mexican limes showed varying expressionlevels of AtFT, which was expected due to positional effect of transgeneinsertion location within the genome. The bars highlighted in pink arethe early-flowering lines. Table 11 further shows the comparison offlowering time of various transgenic Mexican lime lines 70-90 weeksafter transferring from rooting media. Importantly, these transgenicMexican lime lines started to flower as early as 70 weeks aftertransferring to deep soil containers in the greenhouse. Consideringtransgenic citrus lines generated through tissue culture typically need3-5 years before they start flowering, these results indicated that AtFTis able to induce early flowering phenotype.

Phenotypic Analysis of Anthocyanin Accumulation in Transgenic CitrusPlants

The pattern, distribution and level of anthocyanin accumulation in youngimmature transgenic Mexican lime varied widely among different lines anddevelopmental stages. The transgenic lines showed variation in time tofirst flowering but generally appeared to require ˜70 weeks in deep soilcontainers. Anthocyanin accumulation phenotypes of the transgenicPromoter::MoroMybA(MM) Mexican lime lines are shown in FIGS. 16-21.

Results showed that transgenic CitWAXp::MoroMybA Mexican lime (line#9-3) had anthocyanin detected in the midrib of young leaves, flowersmainly in the stigma, style and petals (FIG. 16 and FIG. 17). Whencompared to the WT, fruits of CitWAXp::MoroMybA clearly accumulatedanthocyanin in the young immature fruits mainly in juice sacs, seeds andsegment membranes, but not in flavedo and albedo.

For transgenic CitUNKp::MoroMybA Mexican lime (line #4-3), anthocyaninwas not detected in the young or mature leaves, flowers and but weaklyin the juice sacs/seeds of young immature fruits (FIG. 16 and FIG. 18).

For transgenic CitJuSacp::MoroMybA Mexican lime (line #7-19),anthocyanin was detected in young leaves, not in mature leaves, inflowers mainly in the stigma, style and petals with a visible pinkphenotype (FIG. 16 and FIG. 19). The fruits accumulated anthocyaninstrongly in juice sacs and segment membranes. The flowers showed typicalearly flowering phenotype when FT is overexpressed. Most of thetransgenic flowers developed on leafy inflorescences, apparently inplace of thorns; however, WT usually develops solitary flowers in theaxils of leaves.

Transgenic PfeMybAp::MoroMybA Mexican lime line (#5-4) did not show anyanthocyanin accumulation phenotype in vegetative or immaturereproductive tissues (FIG. 16 and FIG. 20). However, this phenotype isnot unexpected based on the ripening pattern seen in plum where onlymature fruit expressed anthocyanin.

For transgenic E8p::MoroMybA Mexican lime line (#10-11), anthocyanin wasnot detected in the young or mature leaves, flowers or immaturereproductive tissues similar to WT (FIG. 16 and FIG. 21).

When compared to WT, CitWAXp::MoroMybA and CitJuSacp::MoroMybA showedanthocyanin accumulation in juice sacs, seeds and segment membranes, butnot in flavedo and albedo. CitUNKp::MoroMybA showed anthocyaninaccumulation in juice sacs and seeds only (FIG. 16).

No visible phenotypes in flower and fruit were observed for otherPromoter.::MoroMybA transgenic lines.

Conclusion

Taken together, the results of this study indicate that the candidatepromoters were functional in driving expression of MoroMybA in afruit-specific fashion. Anthocyanin accumulation was successfullymodified in transgenic plants transformed with candidate fruit-specificpromoters.

Example 6: Modification of Lycopene Accumulation Using Fruit-SpecificPromoters

The following example illustrates the use of candidate promoters tomodify lycopene accumulation in fruit. During development in plants,there is a coordinated increase in both chlorophyll and carotenoidpigment content, such as the bright red color of lycopene. Increasedlycopene content is usually considered a favorable fruit trait. In thisstudy, three target genes were exploited to increase lycopeneaccumulation.

Methods and Materials

Molecular Constructs

A genetic construct containing the phytoene synthase (PSY) gene drivenby the 35S promoter was generated for expressing PSY in Arabidopsis. Agenetic construct 35S::β/ε cyclase RNAi-35Sp::PSY for silencing filelycopene cyclase as well as overexpressing PSY was generated fortransforming Carrizo citrange plants. A genetic construct having threecitrus fruit promoters (CitJuSacp, CitWAXp and CitUNKp) fused to PSY,RNAi ε-&β-LCY, and DXS, respectively (CitWAXp::RNAiε-&β-LCY-CitJuSacp::PSY-CitUNKp::DXS), together with the FT (floweringgene) construct for increased flowering time, was generated fortransforming Mexican lime plants.

Transgenic Arabidopsis Plants

Agrobacterium tumefaciens strain GV3101 was used for transformation ofArabidopsis ecotype Ler by the floral dip method (Clough and Bent 1998)that is modified by adding 0.01% Silwet L-77 (Lehle Seeds, Round Rock,Tex.) to the infiltration medium. Primary transformants were selected onMS medium (Sigma, St. Louis, Mo.), 1% sucrose, 0.7% agar with 20 μg/mlhygromycin or 50 μg/ml kanamycin as needed for 10 days prior tocultivation in soil.

Trans Genic Citrus Plants

Material used in this study was juvenile shoot of Carrizo citrange andMexican lime plants that had been generated from seed. Technique usedfor transformation is described in de Oliveira et al., HortTechnology(2016) 26(3), 278-286.

Results

Phytoene synthase (PSY) is the first key enzyme in the carotenoidbiosynthetic pathway, and increasing its activity has been shown toincrease the red coloration in other plants. FIG. 22 shows the abilityof this gene to accumulate lycopene in Arabidopsis during fruitdevelopment.

Two enzymes that convert lycopene into carotene in the carotenoidpathway were identified in citrus as ε-LCY and β-LCY. Reducing theexpression of these genes should also increase lycopene accumulation infruit for generation of Cara cara navel-like red-fleshed citruscultivars. FIG. 23 shows the phenotypes of the transgenic Carrizo testlines transformed with the 35S::β/ε cyclase RNAi::PSY construct.

The third target is DXP Synthase (DXS). DXS acts early in the carotenoidpathway to produce initial building blocks for lycopene synthesis.Previous research in plants indicates that the DXS gene is a bottleneckin the system and increasing its production should produce more overalllycopene.

To increase lycopene accumulation in citrus fruits, a genetic constructcombining the above-described targets, together with the FT (floweringgene) construct for increased flowering time, was generated (FIG. 24),where selected candidate citrus fruit promoters were fused to the threetargets (CitWAXp::RNAi ε-&β-LCY-CitJuSacp::PSY-CitUNKp::DXS).

A total of 25 independent transgenic citrus lines were generated.Preliminary analysis of the transgenic Mexican lime callus showed signsof lycopene accumulation as light orange blush (FIG. 25). Preliminaryanalysis of the transgenic Mexican lime and Carrizo young plants showedno visible lycopene accumulation in the vegetative growth of the plants,which is an expected phenotype with the candidate fruit-specificpromoters used. No flowering or fruiting has occurred yet for thesetransgenic citrus lines.

Conclusion

Taken together, the results of this study indicate that the candidatepromoters were functional in driving expression of lycopene pathwaygenes in plants. Preliminary analysis of the transgenic citrus linesshowed expected phenotype in modifying lycopene accumulation in plants.

REFERENCES

-   Blázquez, M. (2007). Quantitative GUS activity assay of plant    extracts. Cold Spring Harbor Protocols, 2007(2), pdb-prot4690.

Collier R, Dasgupta K, Xing Y P, Hernandez B T, Shao M, Rohozinski D,Kovak E, Lin J, de Oliveira M L P, Stover E, McCue K F, Harmon F G,Blechl A, Thomson J G and Thilmony R (2017) Accurate measurement oftransgene copy number in crop plants using droplet digital PCR. Plant J90 (5):1014-1025.

-   Dasgupta K, Thilmony R and Thomson J G (2015) Developing novel Blood    and Cara cara-like citrus varieties. Citrograph 6(3):65-69.-   Dasgupta K, Shao M, Thomson J G (2016) Purple is the new orange—The    development of novel Blood and Cara Cara like citrus varieties.    Citrograph 7(3):54-58.-   Dasgupta K, Thilmony R, Stover E, de Oliveira M L, Thomson J (2017)    Novel R2R3-myb transcription factors from Prunus Americana regulate    differential patterns of anthocyanin accumulation in tobacco and    citrus, G M Crops & Food 81-21.-   de Oliveira M L, Thomson J G, Stover E (2016) High-efficiency    Propagation of Mature ‘Washington Navel’ Orange and Juvenile    ‘Carrizo’ Citrange Using Axillary Shoot Proliferation.    HortTechnology 26(3):278-286.-   de Oliveira M L, Febres V J, Costa M G, Moore G A, Otoni W C (2009)    High-efficiency Agrobacterium-mediated transformation of citrus via    sonication and vacuum infiltration. Plant Cell Rep. 28(3):387-395.-   Jefferson, R A, Kavanagh, T A, and Bevan, M W (1987) GUS fusions:    β-Glucuronidase as a sensitive and versatile gene fusion marker in    higher plants. EMBO J. 6:3901-3907.-   Verde I, Jenkins J, Dondini L, Micali S, Pagliarani G, Vendramin E,    Paris R, Aramini V, Gazza L, Rossini L, Bassi D (2017) The Peach v2.    0 release: high-resolution linkage mapping and deep resequencing    improve chromosome-scale assembly and contiguity. BMC genomics    18(1):225.-   Weigel D, Glazebrook J (2002) Arabidopsis: A Laboratory Manual. Cold    Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

1. A genetic construct comprising a promoter operably linked to aheterologous nucleotide sequence encoding a product of interest, whereinthe promoter comprises a sequence selected from the group consisting ofSEQ ID NOs: 1-5, or a sequence having at least 90% identity thereto. 2.The genetic construct of claim 1, wherein the product of interest is anRNA molecule or a polypeptide.
 3. The genetic construct of claim 1,wherein the product of interest is in a metabolic pathway selected fromthe group consisting of an anthocyanin metabolic pathway, a tocopherolmetabolic pathway, a fatty acid metabolic pathway, a carotenoidmetabolic pathway, a lycopene metabolic pathway, a betalain metabolicpathway, and a flavonoid metabolic pathway.
 4. The genetic construct ofclaim 1, wherein the product of interest is a polypeptide selected fromthe group consisting of a MYB transcription factor, a phytoene synthase(PSY), a lycopene cyclase (LCY), and a DXP synthase (DXS).
 5. Anexpression vector comprising the genetic construct of claim
 1. 6. Atransgenic plant comprising the genetic construct of claim
 1. 7. A plantpart from the transgenic plant of claim 6, wherein the plant partcomprises the genetic construct.
 8. The plant part of claim 7, whereinthe plant part is a stem, a branch, a root, a leaf, a flower, a fruit, aseed, a cutting, a bud, a cell, or a portion thereof.
 9. A method formodifying a fruit phenotype in a plant, comprising: (i) transforming aplant cell with the genetic construct of claim 1, wherein expression ofthe product of interest is associated with modification of the fruitphenotype; (ii) regenerating a plant from the transformed plant cell;and (iii) growing the regenerated plant to produce fruit of the modifiedphenotype.
 10. The method of claim 9, wherein the fruit phenotype isselected from the group consisting of size, weight, color, shape,firmness, glossiness, flavor, aroma, secondary metabolite content, peelthickness, seed number, juice quality, juice sugar content, juice acidcontent, juice taste, juice color, and juice yield.
 11. The method ofclaim 9, wherein the fruit phenotype is selected from the groupconsisting of anthocyanin content, tocopherol content, fatty acidcontent, carotenoid content, lycopene content, betalain content, andflavonoid content.
 12. The method of claim 9, wherein the plant isselected from the group consisting of orange (Citrus sinensis), mandarin(Citrus reticulata), lime (Citrus aurantifolia), grapefruit (Citrusparadisi), lemon (Citrus limon), pomelo (Citrus maxima), citron (Citrusmedico), papeda (Citrus micrantha), and Prunus sp.
 13. A method forcreating a tomato plant with seedless fruit, comprising: (i)transforming a tomato plant cell with the genetic construct of claim 1,wherein the promoter comprises the sequence of SEQ ID NO: 4, or asequence having at least 90% identity thereto; (ii) regenerating atomato plant from the transformed tomato plant cell; and (iii) growingthe regenerated tomato plant to produce seedless fruit.